Compounds for Positron Emission Tomography

ABSTRACT

Described herein are compounds, compositions, and methods for diagnosing and/or monitoring pathogenic disease using positron emission tomography. Also described are conjugates of the formula B-L-P, wherein B is a radical of a targeting agent selected from vitamin receptor binding ligands (such as folate), PSMA binding ligands, or PSMA inhibitors; L is a divalent linker comprising aspartic acid, lysine, or arginine, and P is a radical of an imaging agent or radiotherapy agent, such as a radionuclide or radionuclide containing group, or a radical of a compound capable of binding a radionuclide, such as a metal chelating group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of UnitedStates Provisional Application Ser. Nos. 61/904,387, filed Nov. 14,2013, 61/904,400, filed Nov. 14, 2013, and 61/909,822, filed Nov. 27,2013, the disclosure of each of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The invention described herein pertains to compounds, compositions, andmethods for diagnosing and/or monitoring diseases and disease statesusing radionuclides. In particular, the invention described hereinpertains to compounds, compositions, and methods for diagnosing and/ormonitoring pathogenic disease states using radionuclides for positronemission tomography (PET).

BACKGROUND AND SUMMARY OF THE INVENTION

PET is a nuclear imaging methodology that detects pairs of gamma raysemitted indirectly by a positron-producing radionuclide. Because the twoemitted gamma rays travel in exactly opposite directions, it is possibleto locate their site of origin and thereby reconstruct athree-dimensional image of all positron emitters from a computeranalysis of the origins of emitted gamma rays. Compared to otherradioimaging modalities, such as SPECT, PET reportedly shows highersensitivity (about 2 orders of magnitude), better spatial resolution(about 5 mm), greater signal to noise, and superior tracerquantification in both preclinical and clinical applications. Inaddition, in contrast to the about 90 minutes required for body scansfor a standard SPECT imaging, PET image acquisition may be routinelyperformed in about 20 minutes. Moreover, in vivo PET imaging generallyrequires only subnanomolar (10⁻¹⁰ to 10⁻¹²) concentrations ofradiotracer, which reportedly minimizes potential damage to otherbiological systems. Finally, PET allows for quantitative dynamicimaging, which may facilitate kinetic studies of target engagementthrough receptor occupancy. It has been discovered herein that PETagents may be targeted to predetermined tissues using vitamin receptorsand/or prostate-specific membrane antigen (PSMA).

For example, vitamin receptors are overexpressed on certain pathogeniccells, including many cancer cell types, activated macrophages, andactivated monocytes. In particular, folate receptors are overexpressedin many cancers. The folate receptor, a 38 KD GPI-anchored protein thatbinds the vitamin folic acid with high affinity (<1 nM), isoverexpressed on many malignant tissues, including ovarian, breast,bronchial, and brain cancers. It is estimated that 95% of all ovariancarcinomas overexpress the folate receptor. In contrast, with theexception of kidney, choroid plexus, and placenta, normal tissuesexpress low or nondetectable levels of the folate receptor. Most cellsalso use an unrelated reduced folate carrier to acquire the necessaryfolic acid.

Folate receptors are also overexpressed on activated macrophages, andactivated monocytes. Further, it has also been reported that the folatereceptor β, the nonepithelial isoform of the folate receptor, isexpressed on activated, but not resting, synovial macrophages. Activatedmacrophages can participate in the immune response by nonspecificallyengulfing and killing foreign pathogens within the macrophage, bydisplaying degraded peptides from foreign proteins on the macrophagecell surface where they can be recognized by other immune cells, and bysecreting cytokines and other factors that modulate the function of Tand B lymphocytes, resulting in further stimulation of immune responses.However, activated macrophages can also contribute to thepathophysiology of disease in some instances. For example, activatedmacrophages can contribute to atherosclerosis, rheumatoid arthritis,autoimmune disease states, and graft versus host disease, among otherdisease states.

Following receptor binding of vitamins to vitamin receptors, such asfolic acid and analogs and derivatives of folic acid to folatereceptors, rapid endocytosis delivers the vitamin into the cell, whereit is unloaded in an endosomal compartment at lower pH.

Importantly, covalent conjugation of small molecules, proteins, and evenliposomes to vitamins and other vitamin receptor binding ligands doesnot block the ability of the ligand to bind to its receptor, andtherefore, such ligand conjugates can readily be delivered to and canenter cells by receptor-mediated endocytosis. Accordingly, diagnostic,imaging, and therapeutic agents can be targeted to vitamin receptors,including the folate receptor, for delivery into vitamin receptorexpressing cells.

The prostate is a male reproductive organ that functions to produce andstore seminal fluid, which provides nutrients and fluids for thesurvival of sperm introduced into the vagina during reproduction. Likeother tissues, the prostate gland may develop either malignant(cancerous) or benign (non-cancerous) tumors. Prostate cancer isreportedly one of the most common male cancers in western societies, andis the second leading form of malignancy among American men.

Prostate-specific membrane antigen (PSMA) is a biomarker that isoverexpressed on prostate cancer. PSMA is over-expressed in themalignant prostate tissues when compared to other organs in the humanbody such as kidney, proximal small intestine, and salivary glands. PSMAis also expressed on the neovasculature within many non-prostate solidtumors, including lung, colon, breast, renal, liver and pancreaticcarcinomas, but not on normal vasculature. However, PSMA is expressedminimally in brain. PSMA is a type II cell surface membrane-boundglycoprotein with ˜110 kD molecular weight, including an intracellularsegment (amino acids 1-18), a transmembrane domain (amino acids 19-43),and an extensive extracellular domain (amino acids 44-750). Though thefunctions of the intracellular segment and the transmembrane domains arecurrently reported to be insignificant, the extracellular domain isinvolved in several distinct activities. For example, PSMA plays a rolein the central nervous system, where it metabolizes N-acetyl-aspartylglutamate (NAAG) into glutamic and N-acetyl aspartic acid. PSMA alsoplays a role in the proximal small intestine where it removes γ-linkedglutamate from poly-γ-glutamated folate and α-linked glutamate frompeptides and small molecules.

Though the particular function of PSMA on prostate cancer cells remainsunresolved, PSMA is known to undergo rapid internalization into thecell, similar to cell surface bound receptors like vitamin receptors.PSMA is internalized through clathrin-coated pits and subsequently caneither recycle to the cell surface or go to lysosomes. Accordingly,diagnostic, imaging, and therapeutic agents can be targeted to PSMA fordelivery into PSMA expressing cells, such as prostate cancer cells.

It has been discovered herein that the compounds and compositionsdescribed herein are useful for targeting and delivering radionuclidesfor diagnosing and/or monitoring various diseases and disease statescaused by pathogenic cell populations. In addition, it has beendiscovered that the compounds and compositions described herein are alsouseful for targeting and delivering radionuclides for treating variousdiseases and disease states caused by pathogenic cell populations inradiotherapy.

In one illustrative and non-limiting embodiment of the inventiondescribed herein, compounds and compositions described herein are usedfor diagnosing and/or monitoring, or treating various diseases anddisease states caused by pathogenic cell populations. In anotherillustrative embodiment, methods are described herein for administeringcompounds and compositions described herein for diagnosing and/ormonitoring, or treating various diseases and disease states caused bypathogenic cell populations. In another embodiment, uses of compoundsand compositions are described herein for manufacturing medicaments fordiagnosing and/or monitoring, or treating various diseases and diseasestates caused by pathogenic cell populations. In another embodiment,kits are described herein for preparing and/or using compounds andcompositions described herein for diagnosing and/or monitoring, ortreating various diseases and disease states caused by pathogenic cellpopulations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a postmortem biodistribution study of ¹⁸F-AIF-QC07017 and¹⁸F-AIF-QC07043 folate-NOTA-Al—¹⁸F conjugates in various tissues at 90minutes post injection in nude mice bearing KB tumor xenografts. Foreach tissue, the histogram is in groups of 4 from left to right:¹⁸F-AIF-QC07017, ¹⁸F-AIF-QC07017+excess folic acid, ¹⁸F-AIF-QC07043,¹⁸F-AIF-QC07043+excess folic acid.

FIG. 1B shows a postmortem biodistribution study of ¹⁸F-AIF-QC07017folate-NOTA-Al—¹⁸F conjugate in various tissues at 90 minutes postinjection in nude mice bearing KB tumor xenografts or A549 tumorxenografts. It is to be understood that the vertical axis has beenexpanded and that the kidney data is truncated. For each tissue, thehistogram is in groups of 4 from left to right: ¹⁸F-AIF-QC07017 againstA549 tumor xenografts, ¹⁸F-AIF-QC07017+excess folic acid against A549tumor xenografts, ¹⁸F-AIF-QC07017 against KB tumor xenografts,¹⁸F-AIF-QC07017+excess folic acid against KB tumor xenografts.

FIG. 1C shows a postmortem biodistribution study of ¹⁸F-AIF-QC07043folate-NOTA-Al—¹⁸F conjugate in various tissues at 90 minutes postinjection in nude mice bearing KB tumor xenografts or A549 tumorxenografts. It is to be understood that the vertical axis has beenexpanded and that the kidney data is truncated. For each tissue, thehistogram is in groups of 4 from left to right: ¹⁸F-AIF-QC07043 againstA549 tumor xenografts, ¹⁸F-AIF-QC07043+excess folic acid against A549tumor xenografts, ¹⁸F-AIF-QC07043 against KB tumor xenografts,¹⁸F-AIF-QC07043+excess folic acid against KB tumor xenografts.

FIG. 2A shows a postmortem biodistribution study of ¹⁸F-AIF-QC07017 and¹⁸F-AIF-QC07043 folate-NOTA-Al—¹⁸F conjugates, compared to ^(99m)Tc-EC20in KB tumor xenograft tissues at 90 minutes post injection in nude mice.The histogram from left to right: ^(99m)Tc-EC20 against KB tumorxenografts, ^(99m)Tc-EC20+excess folic acid against KB tumor xenografts,¹⁸F-AIF-QC07017 against KB tumor xenografts, ¹⁸F-AIF-QC07017+excessfolic acid against KB tumor xenografts, ¹⁸F-AIF-QC07043 against KB tumorxenografts, ¹⁸F-AIF-QC07043+excess folic acid against KB tumorxenografts.

FIG. 2B shows a postmortem biodistribution study of ¹⁸F-AIF-QC07017 and¹⁸F-AIF-QC07043 folate-NOTA-Al—¹⁸F conjugates, compared to ^(99m)Tc-EC20in A549 tumor xenograft tissues at 90 minutes post injection in nudemice. The histogram from left to right: ^(99m)Tc-EC20 against A549 tumorxenografts, ^(99m)Tc-EC20+excess folic acid against A549 tumorxenografts, ¹⁸F-AIF-QC07017 against A549 tumor xenografts,¹⁸F-AIF-QC07017+excess folic acid against A549 tumor xenografts,¹⁸F-AIF-QC07043 against A549 tumor xenografts, ¹⁸F-AIF-QC07043+excessfolic acid against A549 tumor xenografts.

DETAILED DESCRIPTION

In each of the foregoing and each of the following embodiments, it is tobe understood that the formulae include and represent not only allpharmaceutically acceptable salts of the compounds, but also include anyand all hydrates and/or solvates of the compound formulae. It isappreciated that certain functional groups, such as the hydroxy, amino,and like groups form complexes and/or coordination compounds with waterand/or various solvents, in the various physical forms of the compounds.Accordingly, the formulae described herein are to be understood toinclude and represent those various hydrates and/or solvates. It is alsoto be understood that the non-hydrates and/or non-solvates of thecompound formulae are described by such formula, as well as the hydratesand/or solvates of the compound formulae.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. It is to beunderstood that the compositions described herein may be prepared fromisolated compounds described herein or from salts, solutions, hydrates,solvates, and other forms of the compounds described herein. It isappreciated that certain functional groups, such as the hydroxy, amino,and like groups form complexes and/or coordination compounds with waterand/or various solvents, in the various physical forms of the compounds.It is also to be understood that the compositions may be prepared fromvarious amorphous, non-amorphous, partially crystalline, crystalline,and/or other morphological forms of the compounds described herein. Itis also to be understood that the compositions may be prepared fromvarious hydrates and/or solvates of the compounds described herein.Accordingly, such pharmaceutical compositions that recite compoundsdescribed herein are to be understood to include each of, or anycombination of, the various morphological forms and/or solvate orhydrate forms of the compounds described herein. In addition, it is tobe understood that the compositions may be prepared from variousco-crystals of the compounds described herein.

Illustratively, compositions may include one or more carriers, diluents,and/or excipients. The compounds described herein, or compositionscontaining them, may be formulated in a therapeutically effective amountin any conventional dosage forms appropriate for the methods describedherein. The compounds described herein, or compositions containing them,including such formulations, may be administered by a wide variety ofconventional routes for the methods described herein, and in a widevariety of dosage formats, utilizing known procedures (see generally,Remington: The Science and Practice of Pharmacy, (21^(st) ed., 2005)).

In each of the foregoing and each of the following embodiments, it isalso to be understood that the formulae include and represent eachpossible isomer, such as stereoisomers and geometric isomers, bothindividually and in any and all possible mixtures. In each of theforegoing and each of the following embodiments, it is also to beunderstood that the formulae include and represent any and allcrystalline forms, partially crystalline forms, and non crystallineand/or amorphous forms of the compounds.

Illustrative embodiments of the invention are described by the followingclauses:

A conjugate of the formula

B-L-P

or a pharmaceutically acceptable salt thereof, wherein B is a radical ofa targeting agent selected from vitamin receptor binding ligands, PSMAbinding ligands, and PSMA inhibitors, L is a divalent linker, and P is aradical of an imaging agent or radiotherapy agent, such as aradionuclide or radionuclide containing group, or a precursor thereof,or a radical of a compound capable of binding a radionuclide orradionuclide containing group, such as a metal chelating group.

The conjugate of the preceding clause wherein the targeting agent is aradical of a folate receptor binding ligand.

The conjugate of any one of the preceding clauses wherein the targetingagent is a radical of a folic acid.

The conjugate of any one of the preceding clauses comprising folate-Asp.

The conjugate of any one of the preceding clauses comprisingfolate-Asp-Arg.

The conjugate of any one of the preceding clauses comprising folate-Arg.

The conjugate of any one of the preceding clauses wherein the linkercomprises a polypeptide.

The conjugate of any one of the preceding clauses wherein the linkercomprises a polypeptide comprising lysine, arginine, or aspartic acid,or a combination thereof.

The conjugate of any one of the preceding clauses wherein the linkercomprises a lysine.

The conjugate of any one of the preceding clauses wherein the linkercomprises Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Arg-Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Arg-Arg-Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Asp-Arg-Arg-Lys.

The conjugate of any one of the preceding clauses wherein the linkerdoes not include a polyamine radical, such as a polyamine diradical ofthe formula NH—(CH₂)₂—NH.

The conjugate of any one of the preceding clauses wherein P comprisesthe formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses comprising the formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses comprising folate-PEG.

The conjugate of any one of the preceding clauses comprisingfolate-PEG₂.

The conjugate of any one of the preceding clauses comprisingfolate-PEG₆.

The conjugate of any one of the preceding clauses comprisingfolate-PEG₁₂.

The conjugate of any one of the preceding clauses wherein the linkercomprises [(CH₂)₂O]_(n), [(CH₂)₂O]_(n)—(CH₂)₂—C(O),[(CH₂)₂O]_(n)—(CH₂)₂—C(O)NH, [(CH₂)₂]_(n)—(CH₂)₂—C(O)NH—(CH₂)₂,[(CH₂)₂O]₂—(CH₂)_(n)—C(O)NH—(CH₂)₂NH, where n is an integer from 1 toabout 12.

The conjugate of any one of the preceding clauses wherein the linkercomprises [(CH₂)₂O]₂, [(CH₂)₂O]₆, or [(CH₂)₂O]₁₂.

The conjugate of any one of the preceding clauses wherein the linkercomprises (CH₂)₂O—(CH₂)₂—C(O), [(CH₂)₂O]₂—(CH₂)₂—C(O),[(CH₂)₂O]₆—(CH₂)₂—C(O), or [(CH₂)₂O]₂—(CH₂)₂—C(O).

The conjugate of any one of the preceding clauses wherein the linkercomprises (CH₂)₂O—(CH₂)₂—C(O)NH, [(CH₂)₂O]₂—(CH₂)₂—C(O)NH,[(CH₂)₂O]₆—(CH₂)₂—C(O)NH, or [(CH₂)₂O]₁₂—(CH₂)₂—C(O)NH.

The conjugate of any one of the preceding clauses wherein the linkercomprises (CH₂)₂O—(CH₂)₂—C(O)NH—(CH₂)₂, [(CH₂)₂O]₂—(CH₂)₂—C(O)NH—(CH₂)₂,[(CH₂)₂O]₆—(CH₂)₂—C(O)NH—(CH₂)₂, or [(CH₂)₂O]₁₂—(CH₂)₂—C(O)NH—(CH₂)₂.

The conjugate of any one of the preceding clauses wherein the linkercomprises (CH₂)₂O—(CH₂)₂—C(O)NH—(CH₂)₂NH,[(CH₂)₂O]₂—(CH₂)₂—C(O)NH—(CH₂)₂NH, [(CH₂)₂O]₆—(CH₂)₂—C(O)NH—(CH₂)₂NH, or[(CH₂)₂O]₁₂—(CH₂)₂—C(O)NH—(CH₂)₂NH.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH[(CH₂)₂O]_(n), NH[(CH₂)₂O]_(n)—(CH₂)₂—C(O),NH[(CH₂)₂O]_(n)—(CH₂)₂—C(O)NH, NH[(CH₂)₂O]_(n)—(CH₂)₂—C(O)NH—(CH₂)₂,NH[(CH₂)₂O]_(n)—(CH₂)₂—C(O)NH—(CH₂)₂NH, where n is an integer from 1 toabout 12.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH(CH₂)₂O, NH[(CH₂)₂O]₂, NH[(CH₂)₂O]₆, NH[(CH₂)₂O]₁₂.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH(CH₂)₂O—(CH₂)₂—C(O), NH[(CH₂)₂O]₂—(CH₂)₂—C(O),NH[(CH₂)₂O]₆—(CH₂)₂—C(O), or NH[(CH₂)₂O]₁₂—(CH₂)₂—C(O).

The conjugate of any one of the preceding clauses wherein the linkercomprises NH(CH₂)₂O—(CH₂)₂—C(O)NH, NH[(CH₂)₂O]₂—(CH₂)₂—C(O)NH,NH[(CH₂)₂O]₆—(CH₂)₂—C(O)NH, or NH[(CH₂)₂O]₁₂—(CH₂)₂—C(O)NH.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH(CH₂)₂O—(CH₂)₂—C(O)NH—(CH₂)₂,NH[(CH₂)₂O]₂—(CH₂)₂—C(O)NH—(CH₂)₂, NH[(CH₂)₂O]₆—(CH₂)₂—C(O)NH—(CH₂)₂, orNH[(CH₂)₂O]₁₂—(CH₂)₂—C(O)NH—(CH₂)₂.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH(CH₂)₂O—(CH₂)₂—C(O)NH—(CH₂)₂NH,NH[(CH₂)₂O]₂—(CH₂)₂—C(O)NH—(CH₂)₂NH,NH[(CH₂)₂O]₆—(CH₂)₂—C(O)NH—(CH₂)₂NH, orNH[(CH₂)₂O]₁₂—(CH₂)₂—C(O)NH—(CH₂)₂NH.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH[(CH₂)₂O]_(n)—(CH₂)₂NH, where n is an integer from 1 toabout 12.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH(CH₂)₂O—(CH₂)₂NH, NH[(CH₂)₂O]₂—(CH₂)₂NH,NH[(CH₂)₂O]₆—(CH₂)₂NH, or NH[(CH₂)₂O]₁₂—(CH₂)₂NH.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH[(CH₂)₂O]_(n)—(CH₂)₂NH—C(O)—(CH₂)₂—C(O), where n is aninteger from 1 to about 12.

The conjugate of any one of the preceding clauses wherein the linkercomprises NH(CH₂)₂O—(CH₂)₂NH—C(O)—(CH₂)₂—C(O),NH[(CH₂)₂O]₂—(CH₂)₂NH—C(O)—(CH₂)₂—C(O),NH[(CH₂)₂O]₆—(CH₂)₂NH—C(O)—(CH₂)₂—C(O), orNH[(CH₂)₂O]₁₂—(CH₂)₂NH—C(O)—(CH₂)₂—C(O).

The conjugate of any one of the preceding clauses comprising the formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses where P comprises theformula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses comprising the formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses wherein the targetingagent is a radical of a PSMA binding ligand or PSMA inhibitor.

The conjugate of any one of the preceding clauses wherein the targetingagent is a radical of a PSMA inhibitor.

The conjugate of any one of the preceding clauses comprising the formula

wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;or

wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;or

where W is O or S.

The conjugate of any one of the preceding clauses wherein the linkercomprises a polypeptide.

The conjugate of any one of the preceding clauses wherein the linkercomprises a polypeptide comprising phenylalanine, lysine, arginine, oraspartic acid, or a combination thereof.

The conjugate of any one of the preceding clauses wherein the linkercomprises a lysine.

The conjugate of any one of the preceding clauses wherein the linkercomprises Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Arg-Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Asp-Arg-Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Arg-Asp-Arg.

The conjugate of any one of the preceding clauses wherein the linkercomprises Arg-Asp-Arg-Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Phe-Arg-Asp.

The conjugate of any one of the preceding clauses wherein the linkercomprises Phe-Arg-Asp-Arg.

The conjugate of any one of the preceding clauses wherein the linkercomprises Phe-Arg-Asp-Arg-Lys.

The conjugate of any one of the preceding clauses wherein the linkercomprises Phe-Phe-Arg.

The conjugate of any one of the preceding clauses wherein the linkercomprises Phe-Phe-Arg-Asp.

The conjugate of any one of the preceding clauses wherein the linkercomprises Phe-Phe-Arg-Asp-Arg.

The conjugate of any one of the preceding clauses wherein the linkercomprises Phe-Phe-Arg-Asp-Arg-Lys.

The conjugate of any one of the preceding clauses wherein the radical ofthe radionuclide or radionuclide containing group, or precursor thereof,or compound capable of binding a radionuclide or radionuclide containinggroup comprises a radical of NOTA.

The conjugate of any one of the preceding clauses wherein P comprisesthe formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses comprising the formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses comprising the formula

wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The conjugate of any one of the preceding clauses comprising the formula

The conjugate of any one of the preceding clauses wherein the linkercomprises the formula

The conjugate of any one of the preceding clauses wherein the linkercomprises the formula

The conjugate of any one of the preceding clauses wherein the linkercomprises the formula

The conjugate of any one of the preceding clauses wherein one or more ofthe phenylalanines is L-phenylalanine.

The conjugate of any one of the preceding clauses comprising the formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses where P comprises theformula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses comprising the formula

or a derivative thereof comprising a chelated metal.

The conjugate of any one of the preceding clauses wherein theradionuclide is a positron emitting radionuclide.

The conjugate of any one of the preceding clauses wherein theradionuclide is a metal ion.

The conjugate of any one of the preceding clauses wherein theradionuclide is a metal salt.

The conjugate of any one of the preceding clauses comprising an aluminumhalide, such as an aluminum fluoride, aluminum chloride, aluminumbromide, or aluminum iodide.

The conjugate of any one of the preceding clauses comprising an aluminumfluoride.

The conjugate of any one of the preceding clauses comprising an aluminum¹⁸F-fluoride.

The conjugate of any one of the preceding clauses comprising an aluminumiodide.

The conjugate of any one of the preceding clauses comprising an aluminum¹²⁵I-iodide.

The conjugate of any one of the preceding clauses comprising a galliumion.

The conjugate of any one of the preceding clauses comprising a ⁶⁶Ga ion.

The conjugate of any one of the preceding clauses comprising a ⁶⁸Ga ion.

The conjugate of any one of the preceding clauses comprising a zirconiumion.

The conjugate of any one of the preceding clauses comprising a ⁸⁹Zr ion.

The conjugate of any one of the preceding clauses comprising a copperion.

The conjugate of any one of the preceding clauses comprising a ⁶⁴Cu ion.

The conjugate of any one of the preceding clauses wherein theradionuclide is a radiotherapy agent, such as iodine, including ¹³¹I,lutetium, including ¹⁷⁷Lu, yttrium, including ⁹⁰Y, strontium, including⁸⁹Sr, samarium, including ¹⁵³Sm, and the like, or a radiotherapy agentcontaining group.

The conjugate of any one of the preceding clauses comprising a lutetiumion, such as a ¹⁷⁷Lu ion.

The conjugate of any one of the preceding clauses comprising a yttriumion, such as a ⁹⁰Y ion.

A conjugate of the formulae

or a pharmaceutically acceptable salt thereof.

A conjugate of the formulae

or a pharmaceutically acceptable salt thereof.

A conjugate of the foimulae

or a pharmaceutically acceptable salt thereof.

A conjugate of the foimulae

or a pharmaceutically acceptable salt thereof.

The conjugate of any one of the preceding clauses wherein P comprisesthe formula

wherein X⁻ is the conjugate base of an acid, such astrifluoromethanesulfonic acid.

The conjugate of any one of the preceding clauses comprising the formula

where X⁻ is a conjugate base of an acid, such astrifluoromethanesulfonic acid.

The conjugate of any one of the preceding clauses wherein P comprisesthe formula

The conjugate of any one of the preceding clauses wherein P comprisesthe formula

The conjugate of any one of the preceding clauses comprising the formula

The conjugate of any one of the preceding clauses comprising the formula

The conjugate of any one of the preceding clauses wherein P comprisesthe formula *NH—C(CH₂OH)₃.

The conjugate of any one of the preceding clauses comprising a boronfluoride.

The conjugate of any one of the preceding clauses comprising a boron¹⁸F-fluoride.

A pharmaceutical composition comprising one or more of the conjugates ofany one of the preceding clauses, in combination with one or morecarriers, diluents, or excipients, or a combination thereof.

A unit dose or unit dosage form composition comprising a diagnosticallyeffective amount of one or more of the conjugates of any one of thepreceding clauses, optionally in combination with one or more carriers,diluents, or excipients, or a combination thereof for diagnosing and/ormonitoring a pathogenic cell population, such as a cancer orinflammatory disease.

A unit dose or unit dosage form composition comprising a therapeuticallyeffective amount of one or more of the conjugates of any one of thepreceding clauses, optionally in combination with one or more carriers,diluents, or excipients, or a combination thereof for treating apathogenic cell population, such as a cancer or inflammatory disease.

A composition for diagnosing and/or monitoring a disease or diseasestate caused at least in part by a pathogenic cell population, such as acancer or inflammatory disease, in a host animal, the compositioncomprising a diagnostically effective amount of one or more of theconjugates of any one of the preceding clauses; or a pharmaceuticalcomposition comprising a diagnostically effective amount of one or moreof the conjugates of any one of the preceding clauses, optionallyfurther comprising one or more carriers, diluents, or excipients, or acombination thereof.

A composition for treating a disease or disease state caused at least inpart by a pathogenic cell population, such as a cancer or inflammatorydisease, in a host animal, the composition comprising a therapeuticallyeffective amount of one or more of the conjugates of any one of thepreceding clauses; or a pharmaceutical composition comprising atherapeutically effective amount of one or more of the conjugates of anyone of the preceding clauses, optionally further comprising one or morecarriers, diluents, or excipients, or a combination thereof.

A method for diagnosing and/or monitoring a disease or disease statecaused at least in part by a pathogenic cell population, such as acancer or inflammatory disease, in a host animal, the method comprisingthe step of administering to the host animal a diagnostically effectiveamount of one or more of the conjugates of any one of the precedingclauses; or a pharmaceutical composition comprising a diagnosticallyeffective amount of one or more of the conjugates of any one of thepreceding clauses, optionally further comprising one or more carriers,diluents, or excipients, or a combination thereof.

A method for treating a disease or disease state caused at least in partby a pathogenic cell population, such as a cancer or inflammatorydisease, in a host animal, the method comprising the step ofadministering to the host animal a therapeutically effective amount ofone or more of the conjugates of any one of the preceding clauses; or apharmaceutical composition comprising a therapeutically effective amountof one or more of the conjugates of any one of the preceding clauses,optionally further comprising one or more carriers, diluents, orexcipients, or a combination thereof.

Use of one or more of the conjugates of any one of the precedingclauses; or a pharmaceutical composition comprising one or more of theconjugates of any one of the preceding clauses, optionally furthercomprising one or more carriers, diluents, or excipients, or acombination thereof, in the manufacture of a medicament for diagnosingand/or monitoring a disease or disease state caused at least in part bya pathogenic cell population, such as a cancer or inflammatory disease,in a host animal.

Use of one or more of the conjugates of any one of the precedingclauses; or a pharmaceutical composition comprising one or more of theconjugates of any one of the preceding clauses, optionally furthercomprising one or more carriers, diluents, or excipients, or acombination thereof, in the manufacture of a medicament for treating adisease or disease state caused at least in part by a pathogenic cellpopulation, such as a cancer or inflammatory disease, in a host animal.

A kit comprising one or more of the conjugates of any one of thepreceding clauses, or a pharmaceutical composition thereof, optionallyfurther comprising one or more carriers, diluents, or excipients, or acombination thereof; an optional solvent; an optional reactioncontainer, and a set of instructions for preparing one or moreradionuclides and combining the one or more radionuclides with the oneor more of the conjugates to prepare an imaging agent, diagnostic agent,or therapeutic agent.

A kit comprising one or more of the conjugates of any one of thepreceding clauses, or a pharmaceutical composition thereof, optionallyfurther comprising one or more carriers, diluents, or excipients, or acombination thereof; an optional solvent; an optional reactioncontainer, and a set of instructions for preparing one or moreradionuclides and combining the one or more radionuclides with the oneor more of the conjugates to prepare an imaging agent, diagnostic agent,or therapeutic agent.

It is to be understood that in each instance where a compound orchemical formula includes an atom or locus that is marked with orincludes a (*), the (*) indicates that the compound or chemical formulais a radical having an open valence at that atom or locus, and that atomor locus is the location for attachment of another radical.

In another illustrative embodiment, the conjugate, composition, unitdose, method, use, or kit of any other embodiment described hereincomprises a compound of formula

or a derivative thereof comprising a chelated metal; or a radical of theforegoing, where each R is in each instance independently selected toform a carboxylic acid or salt thereof, ester, or amide, and R¹, R², andR³, are each independently selected from hydrogen, and alkyl,cycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each ofwhich is optionally substituted.

In another illustrative embodiment, the conjugate, composition, unitdose, method, use, or kit of any other embodiment described hereincomprises 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) or a derivative thereof comprising a chelated metal; or a radicalof the foregoing.

In another illustrative embodiment, the conjugate, composition, unitdose, method, use, or kit of any other embodiment described hereincomprises a compound of formula

or a derivative thereof comprising a chelated metal; or a radical of theforegoing, where each R is in each instance independently selected toform a carboxylic acid or salt thereof, ester, or amide, and R¹, R², andR³, are each independently selected from hydrogen, and alkyl,cycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each ofwhich is optionally substituted, such as the following illustrativecompounds:

R¹ R²

H

H

H

or a carboxylic acid salt or carboxamide derivative (CONH₂) thereof, ora radical of any of the foregoing; or a derivative thereof comprising achelated metal.

In another illustrative embodiment, the conjugate, composition, unitdose, method, use, or kit of any other embodiment described hereincomprises a compound of formula

or a derivative thereof comprising a chelated metal; or a radical of theforegoing, where R⁴ and R⁵ are selected from hydrogen, and alkyl,cycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each ofwhich is optionally substituted, such as the following illustrativecompounds:

R⁴ R⁵

or a carboxylic acid salt or carboxamide derivative (CONH₂) thereof, ora radical of any of the foregoing; or a derivative thereof comprising achelated metal.

In another illustrative embodiment, the conjugate, composition, unitdose, method, use, or kit of any other embodiment described hereincomprises a compound of formula

*H₂C—CO₂H NOTA

or a carboxylic acid salt or carboxamide derivative (CONH₂) thereof, ora radical of any of the foregoing; or a derivative thereof comprising achelated metal.

In another illustrative embodiment, the conjugate, composition, unitdose, method, use, or kit of any other embodiment described hereincomprises a compound selected from the formulae

or a carboxylic acid salt or carboxamide derivative (CONH₂) thereof, ora radical of any of the foregoing, where n is an integer selected from1, 2, 3, 4, 5, or 6; or a derivative thereof comprising a chelatedmetal.

As used herein the Won “radical” generally refers to an open valencecompound or chemical fragment that results after the removal of ahydrogen atom or a hydroxyl group from a carboxylic acid. For example,the following radicals may be formed from L-NETA

where each (*) atom is an open valence for attachment to a linker and/ortargeting agent.

It is to be understood that the foregoing compounds and radicalsthereof, may be further functionalized to attach reactive groups for thesubsequent attachment of linkers and/or targeting groups.Illustratively, the following reactive intermediates are describedherein

where n is 0 or 1, and NX is

and the like.

It is to be understood that the following compounds, and metal chelatesthereof, are not conjugates of the invention:

where n is 1 or 3.

The compounds described herein may contain one or more chiral centers,or may otherwise be capable of existing as multiple stereoisomers. It isto be understood that in one embodiment, the invention described hereinis not limited to any particular sterochemical requirement, and that thecompounds, and compositions, methods, uses, and medicaments that includethem may be optically pure, or may be any of a variety of stereoisomericmixtures, including racemic and other mixtures of enantiomers, othermixtures of diastereomers, and the like. It is also to be understoodthat such mixtures of stereoisomers may include a single stereochemicalconfiguration at one or more chiral centers, while including mixtures ofstereochemical configuration at one or more other chiral centers.Similarly, the compounds described herein may include geometric centers,such as cis, trans, E, and Z double bonds. It is to be understood thatin another embodiment, the invention described herein is not limited toany particular geometric isomer requirement, and that the compounds, andcompositions, methods, uses, and medicaments that include them may bepure, or may be any of a variety of geometric isomer mixtures. It isalso to be understood that such mixtures of geometric isomers mayinclude a single configuration at one or more double bonds, whileincluding mixtures of geometry at one or more other double bonds.

As used herein, the tenn “alkyl” includes a chain of carbon atoms, whichis optionally branched. As used herein, the terms “alkenyl” and“alkynyl” each include a chain of carbon atoms, which is optionallybranched, and include at least one double bond or triple bond,respectively. It is to be understood that alkynyl may also include oneor more double bonds. It is to be further understood that in certainembodiments, alkyl is advantageously of limited length, includingC₁-C₂₄, C₁-C₁₂, C₁-C₆, and C₁-C₄. Illustratively, such particularlylimited length alkyl groups, including C₁-C₈, C₁-C₆, and C₁-C₄ may bereferred to as lower alkyl. It is to be further understood that incertain embodiments alkenyl and/or alkynyl may each be advantageously oflimited length, including C₂-C₂₄, C₂-C₁₂, C₂-C₈, C₂-C₆, and C₂-C₄.Illustratively, such particularly limited length alkenyl and/or alkynylgroups, including C₂-C₈, C₂-C₆, and C₂-C₄ may be referred to as loweralkenyl and/or alkynyl. It is appreciated herein that shorter alkyl,alkenyl, and/or alkynyl groups may add less lipophilicity to thecompound and accordingly will have different phannacokinetic behavior.

In embodiments of the invention described herein, it is to beunderstood, in each case, that the recitation of alkyl refers to alkylas defined herein, and optionally lower alkyl. In embodiments of theinvention described herein, it is to be understood, in each case, thatthe recitation of alkenyl refers to alkenyl as defined herein, andoptionally lower alkenyl. In embodiments of the invention describedherein, it is to be understood, in each case, that the recitation ofalkynyl refers to alkynyl as defined herein, and optionally loweralkynyl. Illustrative alkyl, alkenyl, and alkynyl groups are, but notlimited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl,heptyl, octyl, and the like, and the corresponding groups containing oneor more double and/or triple bonds, or a combination thereof.

As used herein, the term “alkylene” includes a divalent chain of carbonatoms, which is optionally branched. As used herein, the term“alkenylene” and “alkynylene” includes a divalent chain of carbon atoms,which is optionally branched, and includes at least one double bond ortriple bond, respectively. It is to be understood that alkynylene mayalso include one or more double bonds. It is to be further understoodthat in certain embodiments, alkylene is advantageously of limitedlength, including C₁-C₂₄, C₁-C₁₂, C₁-C₈, C₁-C₆, and C₁-C₄.Illustratively, such particularly limited length alkylene groups,including C₁-C₈, C₁-C₆, and C₁-C₄ may be referred to as lower alkylene.It is to be further understood that in certain embodiments alkenyleneand/or alkynylene may each be advantageously of limited length,including C₂-C₂₄, C₂-C₁₂, C₂-C₈, C₂-C₆, and C₂-C₄. Illustratively, suchparticularly limited length alkenylene and/or alkynylene groups,including C₂-C₈, C₂-C₆, and C₂-C₄ may be referred to as lower alkenyleneand/or alkynylene. It is appreciated herein that shorter alkylene,alkenylene, and/or alkynylene groups may add less lipophilicity to thecompound and accordingly will have different pharmacokinetic behavior.In embodiments of the invention described herein, it is to beunderstood, in each case, that the recitation of alkylene, alkenylene,and alkynylene refers to alkylene, alkenylene, and alkynylene as definedherein, and optionally lower alkylene, alkenylene, and alkynylene.Illustrative alkyl groups are, but not limited to, methylene, ethylene,n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene,pentylene, 1,2-pentylene, 1,3-pentylene, hexylene, heptylene, octylene,and the like.

As used herein, the term “linker” includes is a chain of atoms thatconnects two or more functional parts of a molecule to form a conjugate.Illustratively, the chain of atoms is selected from C, N, O, S, Si, andP, or C, N, O, S, and P, or C, N, O, and S. The chain of atomscovalently connects different functional capabilities of the conjugate,such as targeting agents, drugs, diagnostic agents, imaging agents, andthe like. The linker may have a wide variety of lengths, such as in therange from about 2 to about 100 atoms in the contiguous backbone. Theatoms used in forming the linker may be combined in all chemicallyrelevant ways, such as chains of carbon atoms forming alkylene,alkenylene, and alkynylene groups, and the like; chains of carbon andoxygen atoms forming ethers, polyoxyalkylene groups, or when combinedwith carbonyl groups forming esters and carbonates, and the like; chainsof carbon and nitrogen atoms forming amines, imines, polyamines,hydrazines, hydrazones, or when combined with carbonyl groups formingamides, ureas, semicarbazides, carbazides, and the like; chains ofcarbon, nitrogen, and oxygen atoms forming alkoxyamines, alkoxylamines,or when combined with carbonyl groups forming urethanes, amino acids,acyloxylamines, hydroxamic acids, and the like; and many others. Inaddition, it is to be understood that the atoms forming the chain ineach of the foregoing illustrative embodiments may be either saturatedor unsaturated, thus forming single, double, or triple bonds, such thatfor example, alkanes, alkenes, alkynes, imines, and the like may beradicals that are included in the linker. In addition, it is to beunderstood that the atoms forming the linker may also be cyclized uponeach other or be part of cyclic structure to form divalent cyclicstructures that form the linker, including cyclo alkanes, cyclic ethers,cyclic amines, and other heterocycles, arylenes, heteroarylenes, and thelike in the linker. In this latter arrangement, it is to be understoodthat the linker length may be defined by any pathway through the one ormore cyclic structures. Illustratively, the linker length is defined bythe shortest pathway through the each one of the cyclic structures. Itis to be understood that the linkers may be optionally substituted atany one or more of the open valences along the chain of atoms, such asoptional substituents on any of the carbon, nitrogen, silicon, orphosphorus atoms. It is also to be understood that the linker mayconnect the two or more functional parts of a molecule to form aconjugate at any open valence, and it is not necessary that any of thetwo or more functional parts of a molecule forming the conjugate areattached at any apparent end of the linker.

As used herein, the term “cycloalkyl” includes a chain of carbon atoms,which is optionally branched, where at least a portion of the chain incyclic. It is to be understood that cycloalkylalkyl is a subset ofcycloalkyl. It is to be understood that cycloalkyl may be polycyclic.Illustrative cycloalkyl include, but are not limited to, cyclopropyl,cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl,adamantyl, and the like. As used herein, the term “cycloalkenyl”includes a chain of carbon atoms, which is optionally branched, andincludes at least one double bond, where at least a portion of the chainin cyclic. It is to be understood that the one or more double bonds maybe in the cyclic portion of cycloalkenyl and/or the non-cyclic portionof cycloalkenyl. It is to be understood that cycloalkenylalkyl andcycloalkylalkenyl are each subsets of cycloalkenyl. It is to beunderstood that cycloalkyl may be polycyclic. Illustrative cycloalkenylinclude, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl,cycloheptenylpropenyl, and the like. It is to be further understood thatchain forming cycloalkyl and/or cycloalkenyl is advantageously oflimited length, including C₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₅-C₆. It isappreciated herein that shorter alkyl and/or alkenyl chains formingcycloalkyl and/or cycloalkenyl, respectively, may add less lipophilicityto the compound and accordingly will have different pharmacokineticbehavior.

As used herein, the term “heteroalkyl” includes a chain of atoms thatincludes both carbon and at least one heteroatom, and is optionallybranched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur.In certain variations, illustrative heteroatoms also include phosphorus,and selenium. As used herein, the term “cycloheteroalkyl” includingheterocyclyl and heterocycle, includes a chain of atoms that includesboth carbon and at least one heteroatom, such as heteroalkyl, and isoptionally branched, where at least a portion of the chain is cyclic.Illustrative heteroatoms include nitrogen, oxygen, and sulfur. Incertain variations, illustrative heteroatoms also include phosphorus,and selenium. Illustrative cycloheteroalkyl include, but are not limitedto, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

As used herein, the term “aryl” includes monocyclic and polycyclicaromatic carbocyclic groups, each of which may be optionallysubstituted. Illustrative aromatic carbocyclic groups described hereininclude, but are not limited to, phenyl, naphthyl, and the like. As usedherein, the term “heteroaryl” includes aromatic heterocyclic groups,each of which may be optionally substituted. Illustrative aromaticheterocyclic groups include, but are not limited to, pyridinyl,pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl,quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl,benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl,benzisothiazolyl, and the like.

The term “optionally substituted” as used herein includes thereplacement of hydrogen atoms with other functional groups on theradical that is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonicacids and derivatives thereof, carboxylic acids and derivatives thereof,and the like. Illustratively, any of amino, hydroxyl, thiol, alkyl,haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid isoptionally substituted.

As used herein, the teims “optionally substituted aryl” and “optionallysubstituted heteroaryl” include the replacement of hydrogen atoms withother functional groups on the aryl or heteroaryl that is optionallysubstituted. Such other functional groups, also referred to herein asaryl subsituents, illustratively include, but are not limited to, amino,hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl,arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl,nitro, sulfonic acids and derivatives thereof, carboxylic acids andderivatives thereof, and the like. Illustratively, any of amino,hydroxy, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl,arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl,and/or sulfonic acid is optionally substituted.

Illustrative substituents include, but are not limited to, a radical—(CH₂)_(x)Z^(X), where x is an integer from 0-6 and Z^(X) is selectedfrom halogen, hydroxy, alkanoyloxy, including C₁-C₆ alkanoyloxy,optionally substituted aroyloxy, alkyl, including C₁-C₆ alkyl, alkoxy,including C₁-C₆ alkoxy, cycloalkyl, including C₃-C₈ cycloalkyl,cycloalkoxy, including C₃-C₈ cycloalkoxy, alkenyl, including C₂-C₆alkenyl, alkynyl, including C₂-C₆ alkynyl, haloalkyl, including C₁-C₆haloalkyl, haloalkoxy, including C₁-C₆ haloalkoxy, halocycloalkyl,including C₃-C₈ halocycloalkyl, halocycloalkoxy, including C₃-C₈halocycloalkoxy, amino, C₁-C₆ alkylamino, (C₁-C₆ alkyl)(C₁-C₆alkyl)amino, alkylcarbonylamino, N-(C₁-C₆ alkyl)alkylcarbonylamino,aminoalkyl, C₁-C₆ alkylaminoalkyl, (C₁-C₆ alkyl)(C₁-C₆ alkyl)aminoalkyl,alkylcarbonylaminoalkyl, N-(C₁-C₆ alkyl)alkylcarbonylaminoalkyl, cyano,and nitro; or Z^(X) is selected from —CO₂R⁴ and —CONR⁵R⁶, where R⁴, R⁵,and R⁶ are each independently selected in each occurrence from hydrogen,C₁-C₆ alkyl, aryl-C₁-C₆ alkyl, and heteroaryl-C₁-C₆ alkyl.

It is to be understood that in every instance disclosed herein, therecitation of a range of integers for any variable describes the recitedrange, every individual member in the range, and every possible subrangefor that variable. For example, the recitation that n is an integer from0 to 8, describes that range, the individual and selectable values of 0,1, 2, 3, 4, 5, 6, 7, and 8, such as n is 0, or n is 1, or n is 2, etc.In addition, the recitation that n is an integer from 0 to 8 alsodescribes each and every subrange, each of which may for the basis of afurther embodiment, such as n is an integer from 1 to 8, from 1 to 7,from 1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4, etc.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. It is to beunderstood that the compositions described herein may be prepared fromisolated compounds described herein or from salts, solutions, hydrates,solvates, and other forms of the compounds described herein. It isappreciated that certain functional groups, such as the hydroxy, amino,and like groups form complexes and/or coordination compounds with waterand/or various solvents, in the various physical forms of the compounds.It is also to be understood that the compositions may be prepared fromvarious amorphous, non-amorphous, partially crystalline, crystalline,and/or other morphological forms of the compounds described herein. Itis also to be understood that the compositions may be prepared fromvarious hydrates and/or solvates of the compounds described herein.Accordingly, such pharmaceutical compositions that recite compoundsdescribed herein are to be understood to include each of, or anycombination of, the various morphological forms and/or solvate orhydrate forms of the compounds described herein.

Illustratively, compositions may include one or more carriers, diluents,and/or excipients. The compounds described herein, or compositionscontaining them, may be formulated in a diagnostically ortherapeutically effective amount in any conventional dosage formsappropriate for the methods described herein. The compounds describedherein, or compositions containing them, including such formulations,may be administered by a wide variety of conventional routes for themethods described herein, and in a wide variety of dosage formats,utilizing known procedures (see generally, Remington: The Science andPractice of Pharmacy, (21^(st) ed., 2005)).

The term “diagnostically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes diagnosis and/or monitoring of thesymptoms of the disease or disorder being treated. Illustrativediagnostically effective amounts of the conjugate to be administered tothe host animal include about 1 pg/kg to about 10 mg/kg, 1 ng/kg toabout 10 mg/kg, or from about 10 μg/kg to about 1 mg/kg, or from about100 μg/kg to about 500 μg/kg.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known to the researcher, veterinarian, medical doctoror other clinician of ordinary skill. Illustrative therapeuticallyeffective amounts of the conjugate to be administered to the host animalinclude about 1 pg/kg to about 10 mg/kg, 1 ng/kg to about 10 mg/kg, orfrom about 10 μg/kg to about 1 mg/kg, or from about 100 μg/kg to about500 μg/kg.

The term “administering” as used herein includes all means ofintroducing the compounds and compositions described herein to the hostanimal, including, but are not limited to, oral (po), intravenous (iv),intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal,ocular, sublingual, vaginal, rectal, and the like. The compounds andcompositions described herein may be administered in unit dosage formsand/or formulations containing conventional nontoxicpharmaceutically-acceptable carriers, adjuvants, and/or vehicles.

As used herein, the tem) “amino acid” refers generally to beta, gamma,and longer amino acids, such as amino acids of the formula:

—N(R)—(CR′R″)_(q)—C(O)—

where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and q is an integer such as1, 2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from alanine, aspartic acid, asparagine, cysteine,glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine,methionine, proline, glutamine, arginine, serine, threonine, valine,tryptophan, tyrosine, and ornithine, and the like.

It is to be understood that in every instance disclosed herein, therecitation of a range of integers for any variable describes the recitedrange, every individual member in the range, and every possible subrangefor that variable. For example, the recitation that n is an integer from0 to 8, describes that range, the individual and selectable values of 0,1, 2, 3, 4, 5, 6, 7, and 8, such as n is 0, or n is 1, or n is 2, etc.In addition, the recitation that n is an integer from 0 to 8 alsodescribes each and every subrange, each of which may for the basis of afurther embodiment, such as n is an integer from 1 to 8, from 1 to 7,from 1 to 6, from 2 to 8, from 2 to 7, from 1 to 3, from 2 to 4, etc.

In another embodiment, the linkers described herein include a polyether,such as the linkers of the following formulae:

where m is an integer independently selected in each instance from 1 toabout 8; p is an integer selected from 1 to about 10; and n is aninteger independently selected in each instance from 1 to about 3. Inone aspect, m is independently in each instance 1 to about 3.

In another aspect, n is 1 in each instance. In another aspect, p isindependently in each instance about 4 to about 6. Illustratively, thecorresponding polypropylene polyethers corresponding to the foregoingare described herein and may be included in the conjugates as linkers.In addition, it is appreciated that mixed polyethylene and polypropylenepolyethers may be included in the conjugates as linkers. Further, cyclicvariations of the foregoing polyether compounds, such as those thatinclude tetrahydrofuranyl, 1,3-dioxanes, 1,4-dioxanes, and the like aredescribed herein.

In another embodiment, the linkers described herein include a pluralityof hydroxyl functional groups, such as linkers that incorporatemonosaccharides, oligosaccharides, polysaccharides, and the like. It isto be understood that the polyhydroxyl containing linkers comprise aplurality of —(CROH)— groups, where R is hydrogen or alkyl.

In another embodiment, the linkers include one or more of the followingdiradicals:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer from 1to about 3; n1 is an integer from 1 to about 5, or n1 is an integer from2 to about 5, p is an integer from 1 to about 5, and r is an integerselected from 1 to about 3. In one aspect, the integer n is 3 or 4. Inanother aspect, the integer p is 3 or 4. In another aspect, the integerr is 1.

In another embodiment, the linkers include one or more of the followingdiradicals:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer from 1to about 3; n is an integer from 1 to about 5, or from 2 to about 5, pis an integer from 1 to about 5, and r is an integer selected from 1 toabout 3. In one aspect, the integer n is 3 or 4. In another aspect, theinteger p is 3 or 4. In another aspect, the integer r is 1.

In another embodiment, the linker includes one or more of the followingcyclic polyhydroxyl groups:

wherein n is an integer from 2 to about 5, p is an integer from 1 toabout 5, and each r is an independently selected integer from 1 to about4. In one aspect, the integer n is 3 or 4. In another aspect, theinteger p is 3 or 4. In another aspect, each integer r is independently2 or 3. It is understood that all stereochemical forms of such sectionsof the linkers are described herein. For example, in the above formula,the section may be derived from ribose, xylose, glucose, mannose,galactose, or other sugar and retain the stereochemical arrangements ofpendant hydroxyl and alkyl groups present on those molecules. Inaddition, it is to be understood that in the foregoing foiiiiulae,various deoxy compounds are also described. Illustratively, compounds ofthe following formulae are described:

wherein n is equal to or less than r, such as when r is 2 or 3, n is 1or 2, or 1, 2, or 3, respectively.

In another embodiment, the linker includes a polyhydroxyl compound ofthe following formula:

wherein n and r are each an integer selected from 1 to about 3. In oneaspect, the linker includes one or more polyhydroxyl compounds of thefollowing formulae:

It is understood that all stereochemical forms of such sections of thelinkers are described herein. For example, in the above formula, thesection may be derived from ribose, xylose, glucose, mannose, galactose,or other sugar and retain the stereochemical arrangements of pendanthydroxyl and alkyl groups present on those molecules.

In another configuration, the linkers L described herein includepolyhydroxyl groups that are spaced away from the backbone of thelinker. In one embodiment, such carbohydrate groups or polyhydroxylgroups are connected to the back bone by a triazole group, formingtriazole-linked linkers. Illustratively, such linkers include diradicalsof the following formulae:

wherein n, m, and r are integers and are each independently selected ineach instance from 1 to about 5. In one illustrative aspect, m isindependently 2 or 3 in each instance. In another aspect, r is 1 in eachinstance. In another aspect, n is 1 in each instance. In one variation,the group connecting the polyhydroxyl group to the backbone of thelinker is a different heteroaryl group, including but not limited to,pyrrole, pyrazole, 1,2,4-triazole, furan, oxazole, isoxazole, thienyl,thiazole, isothiazole, oxadiazole, and the like. Similarly, divalent6-membered ring heteroaryl groups are described. Other variations of theforegoing illustrative linkers include oxyalkylene groups, such as thefollowing formulae:

wherein n and r are integers and are each independently selected in eachinstance from 1 to about 5; and p is an integer selected from 1 to about4.

In another embodiment, such carbohydrate groups or polyhydroxyl groupsare connected to the back bone by an amide group, forming amide-linkedlinkers. Illustratively, such linkers include diradicals of thefollowing formulae:

wherein each n is an independently selected integer from 1 to about 3,and m is an independently selected integer from 1 to about 22. In oneillustrative aspect, each n is independently 1 or 2. In anotherillustrative aspect, m is selected from about 6 to about 10,illustratively 8. In one variation, the group connecting thepolyhydroxyl group to the backbone of the linker is a differentfunctional group, including but not limited to, esters, ureas,carbamates, acylhydrazones, and the like. Similarly, cyclic variationsare described. Other variations of the foregoing illustrative linkersinclude oxyalkylene groups, such as the following formulae:

wherein n is in each instance an independently selected integer from 1to about 5; and p is an integer selected from 1 to about 4.

In another embodiment, the linkers include one or more of the followingdiradicals:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; each m is anindependently selected integer from 1 to about 3; each n is anindependently selected integer from 1 to about 6, p is an integer from 1to about 5, and r is an integer selected from 1 to about 3. In onevariation, each n is independently 3 or 4. In another variation, theinteger p is 3 or 4. In another variation, the integer r is 1.

In another embodiment, the linkers include one or more of the followingdiradicals:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; each m is anindependently selected integer from 1 to about 3; each n is anindependently selected integer from 2 to about 6, p is an integer from 1to about 5, and r is an integer selected from 1 to about 3. In onevariation, each n is independently 3 or 4. In another variation, theinteger p is 3 or 4. In another variation, the integer r is 1.

In another embodiment, the linkers include one or more of the followingdiradicals:

wherein each m is an independently selected integer from 1 to about 3;each n is an independently selected integer from 1 to about 6, p is aninteger from 1 to about 5, and r is an integer selected from 1 to about3. In one variation, each n is independently 3 or 4. In anothervariation, the integer p is 3 or 4. In another variation, the integer ris 1.

In another embodiment, the linkers include one or more of the followingdiradicals:

wherein each m is an independently selected integer from 1 to about 3;each n is an independently selected integer from 2 to about 6, p is aninteger from 1 to about 5, and r is an integer selected from 1 to about3. In one variation, each n is independently 3 or 4. In anothervariation, the integer p is 3 or 4. In another variation, the integer ris 1.

In another embodiment, the linkers include one or more of the followingdiradicals:

wherein each m is an independently selected integer from 1 to about 3, pis an integer from 1 to about 5, and r is an integer selected from 1 toabout 3. In another variation, the integer p is 3 or 4. In anothervariation, the integer r is 1.

In another embodiment, the linker is a combination of backbone andbranching side motifs such as is illustrated by the following formulae

wherein n is an integer independently selected in each instance from 0to about 3. The above formula are intended to represent 4, 5, 6, andeven larger membered cyclic sugars. In addition, it is to be understoodthat the above formula may be modified to represent deoxy sugars, whereone or more of the hydroxy groups present on the formulae are replacedby hydrogen, alkyl, or amino. In addition, it is to be understood thatthe corresponding carbonyl compounds are described by the aboveformulae, where one or more of the hydroxyl groups is oxidized to thecorresponding carbonyl. In addition, in this illustrative embodiment,the pyranose includes both carboxyl and amino functional groups and (a)can be inserted into the backbone and (b) can provide synthetic handlesfor branching side chains in variations of this embodiment. Any of thependant hydroxyl groups may be used to attach other chemical radicals,including additional sugars to prepare the correspondingoligosaccharides. Other variations of this embodiment are alsodescribed, including inserting the pyranose or other sugar into thebackbone at a single carbon, i.e. a spiro arrangement, at a geminal pairof carbons, and like arrangements. For example, one or two ends of thelinker, or the agent P, or the ligand B may be connected to the sugar tobe inserted into the backbone in a 1,1; 1,2; 1,3; 1,4; 2,3, or otherarrangement.

In another embodiment, the linkers include one or more amino groups ofthe following formulae:

where each n is an integer independently selected in each instance from1 to about 3. In one aspect, the each n is independently 1 or 2 in eachinstance. In another aspect, the integer n is 1 in each instance.

In another embodiment, the linker is a sulfuric acid ester, such as analkyl ester of sulfuric acid. Illustratively, the linker is of thefollowing formula:

where each n is an integer independently selected in each instance from1 to about 3. Illustratively, each n is independently 1 or 2 in eachinstance.

It is understood, that in such polyhydroxyl, polyamino, carboxylic acid,sulfuric acid, and like linkers that include free hydrogens bound toheteroatoms, one or more of those free hydrogen atoms may be protectedwith the appropriate hydroxyl, amino, or acid protecting group,respectively, or alternatively may be blocked as the correspondingpro-drugs, the latter of which are selected for the particular use, suchas pro-drugs that release the parent drug under general or specificphysiological conditions.

It is to be understood that in each of the foregoing illustrativeexamples, the stereochemical configurations shown herein are merelyillustrative, and other stereochemical configurations are described. Forexample in one variation, the corresponding unnatural amino acidconfigurations may be included in the conjugated described herein asfollows:

wherein each n is an independently selected integer from 2 to about 5, pis an integer from 1 to about 5, and r is an integer from 1 to about 4,as described above.

It is to be further understood that in the foregoing embodiments, openpositions, such as (*) atoms are locations for attachment of thetargeting agent B or the agent (P). In addition, it is to be understoodthat such attachment of either or both of B and A may be direct orthrough an intervening linker. Illustrative additional linkers aredescribed in U.S. Pat. No. 7,601,332, the disclosure of which isincorporated herein by reference.

Illustrative bivalent radicals forming part of the linker.

It is to be understood that the bivalent linkers may be combined in anychemically relevant way, either directly or via an interveningheteroatom to construct the linkers described herein.

In another embodiment, the polyvalent linkers described herein comprisea linker selected from the group consisting of carbonyl, thionocarbonyl,alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1 alkylenesuccinimid-3-yl,1 (carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl,alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl.

In another embodiment, the compounds described herein comprise one ormore amino acids.

The compounds described herein can be used for both human clinicalmedicine and veterinary applications. Thus, the host animal harboringthe population of pathogenic cells and administered the compoundsdescribed herein can be human or, in the case of veterinaryapplications, can be a laboratory, agricultural, domestic, or wildanimal. The present invention can be applied to host animals including,but not limited to, humans, laboratory animals such rodents (e.g., mice,rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animalssuch as dogs, cats, and rabbits, agricultural animals such as cows,horses, pigs, sheep, goats, and wild animals in captivity such as bears,pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas,dolphins, and whales.

The compounds, compositions, methods, and uses described herein areuseful for diagnosing and/or monitoring diseases caused at least in partby populations of pathogenic cells, which may cause a variety ofpathologies in host animals. As used herein, the term “pathogenic cells”or “population of pathogenic cells” gernerally refers to cancer cells,infectious agents such as bacteria and viruses, bacteria- orvirus-infected cells, inflammatory cells, activated macrophages capableof causing a disease state, and any other type of pathogenic cells thatuniquely express, preferentially express, or overexpress binding sitesfor the targeting agents described herein.

Illustratively, the population of pathogenic cells can be a cancer cellpopulation that is tumorigenic, including benign tumors and malignanttumors, or it can be non-tumorigenic. The cancer cell population canarise spontaneously or by such processes as mutations present in thegermline of the host animal or somatic mutations, or it can bechemically-, virally-, or radiation-induced. The invention can beutilized to diagnose, monitor, and/or treat such cancers, includingcarcinomas, sarcomas, lymphomas, Hodgekin's disease, melanomas,mesotheliomas, Burkitt's lymphoma, nasopharyngeal carcinomas, leukemias,and myelomas. The cancer cell population can include, but is not limitedto, oral, thyroid, endocrine, skin, gastric, esophageal, laryngeal,pancreatic, colon, bladder, bone, ovarian, cervical, uterine, breast,testicular, prostate, rectal, kidney, liver, and lung cancers.

Illustratively, the population of pathogenic cells can also be activatedmonocytes or macrophages associated with disease states such asfibromyalgia, rheumatoid arthritis, osteoarthritis, ulcerative colitis,Crohn's disease, psoriasis, osteomyelitis, multiple sclerosis,atherosclerosis, pulmonary fibrosis, sarcoidosis, systemic sclerosis,organ transplant rejection (GVHD), lupus erythematosus, Sjogren'ssyndrome, glomerulonephritis, inflammations of the skin, such aspsoriasis, and the like, chronic inflammations, and inflammations due toinjury, such as head or spinal cord injury, embolisms, and the like.

The conjugates described herein can be formed from, for example, a widevariety of vitamins or receptor-binding vitamin analogs/derivatives,linkers, and imaging and radiotherapy agents. The conjugates describedherein are capable of selectively targeting a population of pathogeniccells in the host animal due to preferential expression of a receptorfor the targeting agent, such as a vitamin, accessible for binding, onthe pathogenic cells. Illustrative vitamin moieties that can be used asthe targeting agent (B) include carnitine, inositol, lipoic acid,pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid,riboflavin, thiamine, biotin, vitamin B12, and the lipid solublevitamins A, D, E and K. These vitamins, and their receptor-bindinganalogs and derivatives, constitute an illustrative targeting entitythat can be coupled with the imaging or radiotherapy agent by a bivalentlinker (L) to form a targeting agent (B) imaging or radiotherapy agentconjugate as described herein. The term vitamin is understood to includevitamin analogs and/or derivatives, unless otherwise indicated.Illustratively, pteroic acid which is a derivative of folate, biotinanalogs such as biocytin, biotin sulfoxide, oxybiotin and other biotinreceptor-binding compounds, and the like, are considered to be vitamins,vitamin analogs, and vitamin derivatives. It should be appreciated thatvitamin analogs or derivatives as described herein refer to vitaminsthat incorporates an heteroatom through which the vitamin analog orderivative is covalently bound to the bivalent linker (L).

Illustrative vitamin moieties include folic acid, biotin, riboflavin,thiamine, vitamin B₁₂, and receptor-binding analogs and derivatives ofthese vitamin molecules, and other related vitamin receptor bindingmolecules.

In one embodiment, the targeting group B is a folate, an analog offolate, or a derivative of folate. It is to be understood as usedherein, that the term folate is used both individually and collectivelyto refer to folic acid itself, and/or to such analogs and derivatives offolic acid that are capable of binding to folate receptors.

Illustrative embodiments of vitamin analogs and/or derivatives includefolate and analogs and derivatives of folate such as folinic acid,pteropolyglutamic acid, and folate receptor-binding pteridines such astetrahydropterins, dihydrofolates, tetrahydrofolates, and their deazaand dideaza analogs. The terms “deaza” and “dideaza” analogs refer tothe art-recognized analogs having a carbon atom substituted for one ortwo nitrogen atoms in the naturally occurring folic acid structure, oranalog or derivative thereof. For example, the deaza analogs include the1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folate,folinic acid, pteropolyglutamic acid, and folate receptor-bindingpteridines such as tetrahydropterins, dihydrofolates, andtetrahydrofolates. The dideaza analogs include, for example,1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs offolate, folinic acid, pteropolyglutamic acid, and folatereceptor-binding pteridines such as tetrahydropterins, dihydrofolates,and tetrahydrofolates. Other folates useful as complex forming ligandsfor this invention are the folate receptor-binding analogs aminopterin,amethopterin (also known as methotrexate), N¹⁰-methylfolate,2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin or3-deazamethopterin, and3′,5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid(dichloromethotrexate). The foregoing folic acid analogs and/orderivatives are conventionally termed “folates,” reflecting theirability to bind with folate-receptors, and such ligands when conjugatedwith exogenous molecules are effective to enhance transmembranetransport, such as via folate-mediated endocytosis as described herein.

Additional analogs of folic acid that bind to folic acid receptors aredescribed in US Patent Application Publication Serial Nos. 2005/0227985and 2004/0242582, the disclosures of which are incorporated herein byreference. Illustratively, radicals of such folate analogs have thegeneral formula:

wherein

X and Y are each-independently selected from the group consisting ofhalo, R², OR², SR³, and NR⁴R⁵;

U, V, and W represent divalent moieties each independently selected fromthe group consisting of (R^(6a))C═, N═, (R^(6a))C(R^(7a)), andN(R^(4a));

Q is selected from the group consisting of C and CH;

T is selected from the group consisting of S, O, N, NH, and —C═C—;

A¹ and A² are each independently selected from the group consisting ofoxygen, sulfur, C(Z), C(Z)O, OC(Z), N(R^(4b)), C(Z)N(R^(4b)),N(R^(4b))C(Z), OC(Z)N(R^(4b)), N(R^(4b))C(Z)O, N(R^(4b))C(Z)N(R^(5b)),S(O), S(O)₂, N(R^(4a))S(O)₂, C(R^(6b))(R^(7b)), N(C≡CH), N(CH₂C≡CH),C₁-C₁₂ alkylene, and C₁-C₁₂ alkyeneoxy, where Z is oxygen or sulfur;

R¹ is selected-from the group consisting of hydrogen, halo, C₁-C₁₂alkyl, and C₁-C₁₂ alkoxy; R², R³, R⁴, R^(4a), R^(4b), R⁵, R^(5b),R^(6b), and R^(7b) are each independently selected from the groupconsisting of hydrogen, halo, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₁-C₁₂alkanoyl, C₁-C₁₂ alkenyl, C₁-C₁₂ alkynyl, (C₁-C₁₂ alkoxy)carbonyl, and(C₁-C₁₂ alkylamino)carbonyl;

R⁶ and R⁷ are each independently selected from the group consisting ofhydrogen, halo, C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or, R⁶ and R⁷ are takentogether to form a carbonyl group; R^(6a) and R^(7a) are eachindependently selected from the group consisting of hydrogen, halo,C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or R^(6a) and R^(7a) are taken togetherto form a carbonyl group;

L is one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids;and

n, p, r, s and t are each independently either 0 or 1.

As used herein, it is to be understood that the term folate refers bothindividually to folic acid used in forming a conjugate, or alternativelyto a folate analog or derivative thereof that is capable of binding tofolate or folic acid receptors.

In another embodiment, the targeting group is a PSMA ligand orinhibitor, such as a derivative of pentanedioic acid of the formula:

wherein X is RP(O)(OH)CH₂— (U.S. Pat. No. 5,968,915); RP(O)(OH)N(R¹)—(U.S. Pat. No. 5,863,536); RP(O)(OH)O— (U.S. Pat. No. 5,795,877);RN(OH)C(O)Y— or RC(O)NH(OH)Y, wherein Y is —CR₁R₂—, —NR₃— or —O— (U.S.Pat. No. 5,962,521); RS(O)Y, RSO₂Y, or RS(O)(NH)Y, wherein Y is —CR₁R₂—,—NR₃— or —O— (U.S. Pat. No. 5,902,817); and RS-alkyl, wherein R is forexample hydrogen, alkyl, aryl, or arylalkyl, each of which may beoptionally substituted (J. Med. Chem. 46:1989-1996 (2003)).

In each of the foregoing formulae, R, R₁, R₂, and R₃ are eachindependently selected from hydrogen, C₁-C₉ straight or branched chainalkyl, C₂-C₉ straight or branched chain alkenyl, C₃-C₈ cycloalkyl, C₅-C₇cycloalkenyl, and aryl. In addition, in each case, each of R, R₁, R₂,and R₃ may be optionally substituted, such as with one or more groupsselected from C₃-C₈ cycloalkyl, C₅-C₇ cycloalkenyl, halo, hydroxy,nitro, trifluoromethyl, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy,phenoxy, benzyloxy, amino, aryl. In one aspect, aryl is selected from1-naphthyl, 2-naphthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, benzyl, andphenyl, and in each case aryl may be optionally substituted with one ormore, illustratively with one to three, groups selected from halo,hydroxy, nitro, trifluoromethyl, C₁-C₆ straight or branched chain alkyl,C₂-C6 straight or branched chain alkenyl, C₁-C₄ alkoxy, C₂-C₄alkenyloxy, phenoxy, benzyloxy, and amino. In one variation of each ofthe above formulae, R is not hydrogen.

Illustrative PSMA ligands (U.S. Pat. No. 5,968,915) include2-[[methylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[phenylhydroxyphosphinyl]methyl]pentanedioic acid;2-[[2-(tetrahydrofuranyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[((4-pyridyl)methyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[((2-pyridyl)methyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[(phenylmethyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl] pentanedioic acid;2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]pentanedioic acid; and2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic acid.

Illustrative PSMA ligands (U.S. Pat. No. 5,863,536) includeN-[methylhydroxyphosphinyl]glutamic acid;N-[ethylhydroxyphosphinyl]glutamic acid;N-[propylhydroxyphosphinyl]glutamic acid;N-[butylhydroxyphosphinyl]glutamic acid;N-[phenylhydroxyphosphinyl]glutamic acid;N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid;N-[((phenylethyl)methyl)hydroxyphosphinyl]glutamic acid; andN-methyl-N-[phenylhydroxyphosphinyl]glutamic acid.

Illustrative PSMA ligands (U.S. Pat. No. 5,795,877) include2-[[methylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[ethylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[propylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[butylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[phenylhydroxyphosphinyl]oxy]pentanedioic acid;2-[[((4-pyridyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid;2-[[((2-pyridyl)methyl)hydroxyphosphinyl]oxy]pentanedioic acid;2-[[(phenylmethyl)hydroxyphosphinyl]oxy]pentanedioic acid; and2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy] pentanedioic acid.

Illustrative PSMA ligands (U.S. Pat. No. 5,962,521) include2-[[(N-hydroxy)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-methyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-butyl-N-hydroxy) carbamoyl]methyl]pentanedioic acid;2-[[(N-benzyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-phenyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-2-phenylethyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-ethyl-N-hydroxy) carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-propyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-3-phenylpropyl)carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy-N-4-pyridyl) carbamoyl]methyl]pentanedioic acid;2-[[(N-hydroxy)carboxamido]methyl]pentanedioic acid; 2-[[N-hydroxy(methyl) carboxamido]methyl]pentanedioic acid; 2-[[N-hydroxy (benzyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(phenyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(2-phenylethyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(ethyl)carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy(propyl) carboxamido]methyl]pentanedioic acid;2-[[N-hydroxy (3-phenylpropyl) carboxamido]methyl]pentanedioic acid; and2-[[N-hydroxy(4-pyridyl)carboxamido]methyl]pentanedioic acid.

Illustrative PSMA ligands (U.S. Pat. No. 5,902,817) include2-[(sulfinyl)methyl]pentanedioic acid;2-[(methylsulfinyl)methyl]pentanedioic acid;2-[(ethylsulfinyl)methyl]pentanedioic acid;2-[(propylsulfinyl)methyl]pentanedioic acid;2-[(butylsulfinyl)methyl]pentanedioic acid;2-[(phenylsulfinyl]methyl]pentanedioic acid;2-[[(2-phenylethyl)sulfinyl]methyl]pentanedioic acid;2-[[(3-phenylpropyl)sulfinyl]methyl]pentanedioic acid;2-[[(4-pyridyl)sulfinyl]methyl]pentanedioic acid;2-[(benzylsulfinyl)methyl]pentanedioic acid;2-[(sulfonyl)methyl]pentanedioic acid;2-[(methylsulfonyl)methyl]pentanedioic acid;2-[(ethylsulfonyl)methyl]pentanedioic acid;2-[(propylsulfonyl)methyl]pentanedioic acid;2-[(butylsulfonyl)methyl]pentanedioic acid;2-[(phenylsulfonyl)methyl]pentanedioic acid;2-[[(2-phenylethyl)sulfonyl]methyl]pentanedioic acid;2-[[(3-phenylpropyl)sulfonyl]methyl]pentanedioic acid; 2-[[(4-pyridyl)sulfonyl]methyl]pentanedioic acid;2-[(benzylsulfonyl)methyl]pentanedioic acid;2-[(sulfoximinyl)methyl]pentanedioic acid;2-[(methylsulfoximinyl)methyl]pentanedioic acid;2-[(ethylsulfoximinyl)methyl]pentanedioic acid;2-[(propylsulfoximinyl)methyl]pentanedioic acid;2-[(butylsulfoximinyl)methyl]pentanedioic acid;2-[(phenylsulfoximinyl)methyl]pentanedioic acid;2-[[(2-phenylethyl)sulfoximinyl]methyl]pentanedioic acid;2-[[(3-phenylpropyl) sulfoximinyl]methyl]pentanedioic acid;2-[[(4-pyridyl)sulfoximinyl]methyl]pentanedioic acid; and2-[(benzylsulfoximinyl)methyl]pentanedioic acid.

Illustrative PSMA ligands include

In another embodiment, the PSMA ligand is a urea of two amino acids. Inone aspect, the amino acids include one or more additional carboxylicacids. In another embodiment, the amino acids include one or moreadditional phosphoric, phosphonic, phosphinic, sulfinic, sulfonic, orboronic acids. In another aspect, the amino acids include one or morethiol groups or derivatives thereof. In another aspect, the amino acidsinclude one or more carboxylic acid bioisosteres, such as tetrazoles andthe like.

In another embodiment, the PSMA ligand is a compound of the formula:

-   -   where R¹ is

In another illustrative embodiment, the binding agent is a urea of anamino dicarboxylic acid, such as aspartic acid, glutamic acid, and thelike, and another amino dicarboxylic acid, or an analog thereof, such asa binding agent of the formulae

wherein Q is a an amino dicarboxylic acid, such as aspartic acid,glutamic acid, or an analog thereof, n and m are each independentlyselected from an integer between 1 and about 6, and (*) represents thepoint of attachment for the linker L.

Illustratively, the PSMA ligand is a compound of the formulae:

In another embodiment, the PSMA ligand is2-[3-(1-Carboxy-2-mercapto-ethyl)-ureido]-pentanedioic acid (MUPA) or2-[3-(1,3-Dicarboxy-propyl)-ureido]-pentanedioic acid (DUPA).

Other illustrative examples of PSMA ligands include peptide analogs suchas quisqualic acid, aspartate glutamate (Asp-Glu), Glu-Glu, Gly-Glu,γ-Glu-Glu, beta-N-acetyl-L-aspartate-L-glutamate (β-NAAG), and the like.

In another embodiment, the PSMA ligand comprises a urea or thiourea oflysine and an amino acid, or one or more carboxylic acid derivativesthereof, including, but not limited to ureas or thioureas of lysine andaspartic acid, or glutamic acid, or homoglutamic acid.

In another embodiment, the PSMA ligand comprises a urea or thiourea ofL-lysine and L-glutamate.

In another embodiment, the PSMA ligand comprises a compound selectedfrom the following

In another embodiment, the PSMA ligand comprises the following

The compounds, linkers, intermediates, and conjugates described hereinmay be prepared using conventional processes, including the described inInternational Patent Publication Nos. WO 2009/002993, WO 2004/069159, WO2007/022494, and WO 2006/012527, and U.S. patent application Ser. No.13/837,539 (filed Mar. 15, 2013). The disclosures of each of theforegoing are herein incorporated by reference in their entirety.

Each publication cited herein is incorporated herein by reference.

In another embodiment, a method is described for diagnosing and/ormonitoring a disease or disease state where the method comprises thesteps of administering to a patient being evaluated for the diseasestate an effective amount of a conjugate of the general formula B-L-P.The method includes allowing sufficient time for the conjugate to bindto the target tissue, and diagnosing and/or monitoring the disease ordisease state extra-corporeally, such as by using positron emissiontomography.

The radionuclide may include a positron-emitting isotope having asuitable half-life and toxicity profile. In various embodiments, theradioisotope has a half-life of more than 30 minutes, more than 70minutes, more than 80 minutes, more than 90 minutes, more than 100minutes, less than 8 hours, less than 6 hours, less than 4 hours, orless than 3 hours. In other embodiments, the radioisotope has ahalf-life of about 30 minutes to about 4 hours, about 70 minutes toabout 4 hours, about 80 minutes to about 4 hours, about 90 minutes toabout 4 hours, about 100 minutes to about 4 hours, about 30 minutes toabout 6 hours, about 70 minutes to about 6 hours, about 80 minutes toabout 6 hours, about 90 minutes to about 6 hours, about 100 minutes toabout 6 hours, about 30 minutes to about 8 hours, about 70 minutes toabout 8 hours, about 80 minutes to about 8 hours, about 90 minutes toabout 8 hours, or about 100 minutes to about 8 hours.

The radionuclide may include one or more positron-emitting isotopes,such as but not limited to isotopes selected from ⁸⁹Zr ⁴⁵Ti, ⁵¹Mn, ⁶⁴Cu,⁶¹Cu, ⁶³Zn, ⁸²Rb, ⁶⁸Ga, ⁶⁶Ga, ¹¹C, ¹³N, ¹⁵O, ¹²⁴I, ³⁴Cl, and ¹⁸F. Inanother embodiment, the radionuclide is a halide, such as apositron-emitting halide. In another embodiment, the radionuclide is ametal ion, such as a positron-emitting metal ion. In another embodiment,the radionuclide is a gallium ion, such as a positron-emitting galliumion. In another embodiment, the radionuclide is selected from ⁸⁹Zr,⁶⁴Cu, ⁶⁸Ga, ⁶⁶Ga, ¹²⁴I, and ¹⁸F. In another illustrative embodiment, theradioisotope is selected from ⁸⁹Zr, ⁶⁴Cu, ⁶⁸Ga, ¹²⁴I, and ¹⁸F. Inanother embodiment, the radioisotope is ⁶⁸Ga, or ⁸⁹Zr, or ¹⁸F. Inanother embodiment in each of the foregoing and following embodimentsdescribed herein, the radioisotope is ⁶⁸Ga. In another embodiment ineach of the foregoing and following embodiments described herein, theradioisotope is ¹⁸F. In another embodiment in each of the foregoing andfollowing embodiments described herein, the radioisotope is ⁸⁹Zr. Inanother embodiment in each of the foregoing and following embodimentsdescribed herein, the radioisotope is ⁶⁴Cu. It is also to be understoodthat the fluorine isotopes described herein may be selected from variousisotopic combinations of ¹⁸F and ¹⁹F. It is understood that factors thatmay be included during selection of a suitable isotope includesufficient half-life of the positron-emitting isotope to permitpreparation of a diagnostic composition in a pharmaceutically acceptablecarrier prior to administration to the patient, and sufficient remaininghalf-life to yield sufficient activity to permit extra-corporealmeasurement by a PET scan. Further, a suitable isotope should have asufficiently short half-life to limit patient exposure to unnecessaryradiation. In an illustrative embodiment, ¹⁸F, having a half-life of 110minutes, provides adequate time for preparation of the diagnosticcomposition, as well as an acceptable deterioration rate. Further, ondecay ¹⁸F is converted to ¹⁸O.

Illustrative positron-decaying isotopes having suitable half-livesinclude ³⁴Cl, half-life about 32 minutes; ⁴⁵Ti, half-life about 3 hours;⁵¹Mn, half-life about 45 minutes; ⁶¹Cu, half-life about 3.4 hours; ⁶³Zn,half-life about 38 minutes; ⁸²Rb, half-life about 2 minutes; ⁶⁸Ga,half-life about 68 minutes, ⁶⁶Ga, half-life about 9.5 hours, ¹¹C,half-life about 20 minutes, ¹⁵O, half-life about 2 minutes, ¹³N,half-life about 10 minutes, or ¹⁸F, half-life about 110 minutes.

In another embodiment, the radionuclide is a radiotherapy agent.Illustrative radionuclides for radiotherapy include isotopes of lutetiumsuch as ¹⁷⁷Lu, isotopes of yttrium, such as ⁹⁰Y, isotopes of copper,such as ⁶⁷Cu and ⁶⁴Cu, and the like.

The radionuclide may be covalently attached to the conjugate, such as toan aryl or heteroaryl aromatic group, including benzamidyl, benzylic,phenyl, pyridinyl, pyrimidinyl, pyridazinyl, naphthyl, benzothiazolyl,benzimizolyl, benzoxazolyl, and like groups. In one illustrativeembodiment, the radioisotope is ¹⁸F and the radionuclide includes anaryl group to which the radioisotope is covalently attached.

The radionuclide may be non-covalently attached to the conjugate, suchas within a chelate.

The methods may also be used in combination with any other methods ofcancer diagnosis already developed and known in the art, includingmethods using other already developed diagnostic agents and utilizingx-ray computed tomography (CT), magnetic resonance imaging (MRI),functional magnetic resonance imaging (fMRI), ultrasound, and singlephoton emission computed tomography (SPECT).

It is understood that in certain applications of the methods describedherein, each of the processes and synthetic methods described hereineither substantially complete fluorination, or alternatively onlypartial fluorination may be desired. Accordingly, the processes andsynthetic methods described herein may be performed in variousalternative embodiments. It is therefore understood that in thoseaspects where only partial fluorination is desired, the processes andsyntheses described herein may be performed with less thanstoichiometric amounts of fluorinating agent. Similarly, it isunderstood that in certain applications of the methods described herein,each of the processes and synthetic methods described herein eithersubstantially complete radiofluorination, or alternatively only partialradiofluorination may be desired. Accordingly, the processes andsynthetic methods described herein may be performed in variousalternative embodiments. It is therefore understood that in thoseaspects where only partial radiofluorination is desired, the processesand syntheses described herein may be performed with less thanstoichiometric amounts of radiofluorination agent, where the balance isoptionally ¹⁹F.

The following examples further illustrate specific embodiments of theinvention; however, the following illustrative examples should not beinterpreted in any way to limit the invention.

EXAMPLES

General. Water was distilled and then deionized (18 MΩ/cm2) by passingthrough a Milli-Q water filtration system (Millipore Corp., Milford,Mass.). All chemicals and solvents, unless specified, were purchasedfrom Sigma (St. Louis, Mo.) and were used without further purificationAmino acids were purchased from Chem-Impex Int (Chicago, Ill.).2,2′-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diaceticacid (NOTA-NHS) was purchased from CheMatech (France). N10-TFA-PteroicAcid was provided by Endocyte, Inc. High-performance liquidchromatography (HPLC) analysis and purification of the DUPA-NOTAprecursor were performed on an Agilent G6130B instrument. Theradioactive HPLC was performed with a γ-counter using a Xselect CSH C18(250×10 mm) column and MeCN and 0.1% Formic Acid as mobile phases.

EXAMPLE. C-NETA. tert-Butyl [2-Hydroxy-1-(4-nitrobenzyl)ethyl]carbamate(QC04011) was prepared from the commercially available methyl2-amino-3-(4-nitrophenyl)propanoate through NaBH₄ reduction andBoc-protection. Successive Dess-Martin oxidation and reductive aminationwith QC04001 afforded tris-Boc protected compound QC04013, which wastransformed to QC04014 after Boc-deprotection in 4 M HCl in dioxane.Treatment of QC04014 with tert-butyl bromoacetate, followed byhydrogenolysis of the NO₂ group provided QC04016. Further reaction ofQC04016 with succinic anhydride provided the bifunctional C-NETA(QC04018) as the corresponding tert-butyl ester.

EXAMPLE. Di-tert-butyl [1,4,7]Triazanonane-1,4-dicarboxylate (QC04001).QC04001 was prepared according to a modification of a syntheticprocedure reported previously.[19-21] To a solution of 1,4,7-triazonanetrihydrogenchloride (TACN3.HCl, 1.85 g, 7.7 mmol, M.W.: 238.6) in CHCl₃(25 mL) was added DIPEA (4.0 mL, 3.0 g, 23.1 mmol, M.W.: 129.24, d:0.742) and BOC—ON (3.77 g, 15.3 mmol, M.W.: 246.26) in portions. Theresulting mixture was stirred for 5 days and the solvent evaporatedunder vacuum. The residue was partitioned between 10% NaOH solution (10mL) and diethyl ether (30 mL). The ether layer was separated and washedwith 10% NaOH solution (10 mL) and water (10 mL) several times. Theether layer was dried (MgSO₄), filtered, and concentrated under vacuumto provide QC04001 (2.53 g, quantitative), which was used without furherpurification. ¹H NMR (400 MHz, CDCl₃) δ=3.47-3.50 (m, 2H), 3.42-3.45 (m,2H), 3.38 (br, s, 1H), 3.28-3.34 (m, 2H), 3.16-3.28 (m, 2H), 2.86-2.99(m, 4H), 1.48 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ=156.08, 155.85 (C═O),79.80, 79.70 (^(t-)Bu), 53.20, 52.62, 52.52, 51.78, 50.50, 49.91, 49.63,48.39, 48.23, 47.83, 47.46 (TACN ring from 53.20-47.46), 28.60(^(t-)Bu).

EXAMPLE. tert-Butyl [2-Hydroxy-1-(4-nitrobenzyl)ethyl]carbamate(QC04011)[19]. With minor revision to the reported procedure,[19] wherethe HCl salt of methyl 2-amino-3-(4-nitrophenyl)propanoate was useddirectly without neutralization with Et3N, to a solution of the methyl2-amino-3-(4-nitrophenyl)propanoate hydrochloride salt (6.22 g, 23.9mmol) in MeOH (70 mL) at 23° C. was added NaBH₄ (2.86 g, 71.4 mmol) inmultiple portions. The reaction was monitored by TLC and LC-MS. Themixture was heated to reflux (with water bath at ˜70° C.), and NaBH₄ wasadded portion-wise as needed until most of the starting materialdisappeared, requiring about 6 grams of NaBH4 in total. Afterevaporation of the solvent, the residue was treated with H₂O (70 mL) andextracted with DCM/IPA (3/1). The combined organic layer was dried,filtered, and concentrated under vacuum to provide white solid QC04010(4.4 g, 94), which was used without further purification.

EXAMPLE. QC04010 (4.4 g, 22.7 mmol) was dissolved in CH₃CN (30 mL) atambient temperature, to which was added BOC—ON (11.2 g, 27.2 mmol, 1.2eq.) portionwise. To the above mixture was added DIPEA (5.24 mL, 3.76 g,29.2 mmol, M.W.: 129.24, d: 0.742), the resulting mixture was stirredfor 4 h and evaporated. The residue was partitioned between ether (50mL) and 10% NaOH solution (20 mL). The ether layer was separated andwashed with 10% NaOH solution (10 mL) and water (10 mL) sequentially.The ether layer was dried, filtered, and concentrated under vacuum. Theresidue was washed with ether (20 mL) to provide QC04011 (5.31 g, 75%),which was used without further purification. To prepare an analyticalsample, the residue is purified via column chromatography on SiO₂eluting with Hexane/Ethyl Acetate (3/1 to 1/1 with 1% of MeOH) to affordpure QC04011 as a white solid. ¹H NMR (400 MHz, CDCl₃) δ=8.15 (d, J=8.8MHz, 2H), 7.40 (d, J=8.8 MHz, 2H), 4.84 (d, J=6.8 MHz, 1H), 3.90 (s,1H), 3.68 (dd, J=3.1 MHz, 1H), 3.57 (dd, J=3.1 MHz, 1H), 2.98 (d, J=6.0MHz, 2H), 1.39 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ=156.0, 146.4, 146.2,130.1, 123.5, 79.8, 63.3, 53.1, 37.3, 28.0.

EXAMPLE. tert-Butyl (1-(4-nitrophenyl)-3-oxopropan-2-yl)carbamate.QC04011 (1.27 g, 4.3 mmol) was dissolved in CH₂Cl₂ (40 mL), and cooledto 0° C., to which Dess-Martin periodinane (1.70 g, 5.16 mmol, 1.2equiv) was added in one portion. After stirring for 15 min at 0° C., thereaction was warmed to 23° C. and stirred for 45 min. The reaction wasquenched by addition of a basic aq Na₂S₂O₃ solution (50/50, v/v of aqNa₂S₂O₃ and aq Na₂HCO₃), and the resulting mixture was vigorouslystirred for 15 min. After extraction with CH₂Cl₂ (3×), the organicphases were washed successively with water and brine, dried over Na₂SO₄,filtered and concentrated in vacuo to provide QC04012, which was usedwithout further purification.

EXAMPLE. Reductive amination of QC04012 and QC04001 to prepareQC04013:⁴. 1,4-Di-tert-butyl 7-(2-{[(tert-Butoxy)carbonyl]amino}3-(4-nitrophenyl) propyl)-1,4,7-triazonane-1,4-dicarboxylate (QC04013):Compound QC04012 (4.3 mmol in theory) was added to a solution of QC04001(1.40 g, 4.3 mmol) in DCE (100 mL) at 0° C. The resulting solution wasstirred for 10 min and sodium triacetoxyborohydride (1.28 g, 6.02 mmol,1.4 eq.) was added portionwise to the solution over 30 min The mixturewas stirred at ambient temperature overnight. The reaction mixture wasconcentrated, treated with a saturated aqueous solution of NaHCO₃ (50mL), and extracted with ethyl acetate (3×50 mL). The combined organiclayers were dried with Na₂SO₄, filtered, and concentrated in vacuo. Theresidue was purified via flash chromatography (SiO₂, Hex/EA=3/1) toprovide QC04013 (2.31 g, 88.5% for 2 steps, based on 2.61 g in theory)as a pale yellow semi-solid. ¹H NMR (400 MHz, CDCl₃) δ=8.11 (2H, d,J=7.6 Hz), 7.35 (2H, d, J=7.6 Hz), 5.28 (1H, s, br), 3.54-3.88 (2H, m),3.39-3.54 (2H, m), 3.32-3.40 (1H, m), 3.15-3.32 (2H, m), 2.79-3.15 (4H,m), 2.37-2.73 (6H, m), 1.43 (9 H, s), 1.42 (9 H, s), 1.38 (9 H, s); ¹³CNMR (101 MHz, CDCl₃) δ=156.15, 155.99, 155.70, 155.56, 147.00, 146.95,146.81, 146.76, 130.36, 123.73, 123.65, 123.60, 80.07, 79.99, 79.92,79.81, 79.57, 79.46, 60.79, 60.47, 55.52, 54.33, 54.06, 53.64, 53.15,53.28, 51.54, 50.80, 50.71, 50.42, 49.87, 49.07, 48.12, 39.67, 39.45,28.74, 28.61. MS m/z: MS-API: Calcd. for C₃₀H₅₀N₅O₈ ([M+H]⁺): 608.4,Found: 608.3;

EXAMPLE. 1-(4-Nitrophenyl)-3-(1,4,7-triazonan-1-yl)propan-2-amine.QC04013 (2.31 g, 3.8 mmol) was dispersed in 30 mL of 4 M HCl/Dioxane,the resulting mixture was stirred at room temperature for 20 hours. Thereaction mixture was rapidly added to cold Et₂O to precipitate a whitesolid. The solid was collected and dried in air to afford the pureproduct QC04014 (1.71 g, in quantitative yield) as a pale-white solid.MS m/z: MS-API: Calcd. for C₁₅H₂₆N₅O₂([M+H]⁺): 308.2, Found: 308.2;

EXAMPLE. Introduction of the tri-tert-butyl ethylacetate^(1b). To asolution of QC04014 (78 mg, 0.19 mmol) and DIPEA (0.272 mL, 202 mg, 1.56mmol, 8.2 eq. M.W.: 129.24, d: 0.742) in DMF (2 mL) was added NaI (233.8mg, 1.56 mmol, 8.2 eq. M.W.: 149.89) and tert-Butyl bromoacetate (0.126mL, 168 mg, 0.86 mmol, 4.5 eq. M.W.: 195.05, d: 1.321) slowly at roomtemperature. The resulting mixture was warmed to 60-70° C. and stirredfor 20 hs. After completion, monitored by TLC and LC-MS, the reactionwas quenched by water and extracted with Et₂O. The combined organicsolvent was washed successively with water and brine, and dried overNa₂SO₄. After filtration, the solvent was evaporated under vacuum, andresulting deep-colored oil residue was purified by flash chromatographyon SiO₂ (DCM/MeOH=100/1-100/4) to provide QC04015 (14 mg, 10%) as ayellow oil and QC04015′ (61 mg, 49.4%). MS m/z: MS-API: Calcd. forC₃₉H₆₆N₅O₁₀ ([M+H]⁺): 764.5, Found: 764.4;

EXAMPLE. To a solution of QC04015 (20 mg, 0.039 mmol) in MeOH (2 mL) wasadded 10% Pd/C catalyst (5 mg). The resulting mixture was subjected tohydrogenolysis by agitation with H₂ (g) at 1 atm (˜15 psi) at ambienttemperature for 14 h. The reaction mixture was diluted with excess DCMand filtered through celite, and the filtrate was concentrated in vacuoto provide QC04016 (13 mg, 67.5%). MS m/z: MS-API: Calcd. for C₃₉H₆₈N₅O₈([M+H]⁺): 734.5, Found: 734.4;

FOLATE TARGETED EXAMPLES

EXAMPLE. (S)-5-tert-butyl 1-methyl2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)pentanedioate(QCO2023). HCl.H₂N-Glu(O^(t)Bu)-OMe (350 mg, 1.38 mmol) was added to asolution of N¹⁰-TFA-Pteroic Acid (560 mg, 1.37 mmol) and DIPEA (1.2 mL,6.85 mmol) in DMSO (6.0 mL) at 23° C. under N₂. After stirring for 15min at 23° C., PyBOP (720 mg, 1.0 mmol) was added, and the reactionmixture was stirred for 24 h at 23° C. Volatile material was removedunder reduced vacuum to afford the crude product as a semi-solid, whichwas further purified via solid extraction with Hex/EA (1/1) 3 times toprovide QCO2023 as a pale-yellow solid in quantitative yield, which wasused without further purification. λ_(max)=280 nm; LC-MS (Agilent G6130BQuadrupole LC/MS): Mobile phase: Buffer (pH 7)-CH3CN; Column: AnalyticC18 column; Method: 0-100 CH3CN-15 min, t_(R)=5.62 min. MS m/z: MS-API:Calcd. for C₂₆H₂₉F₃N₇O₇ ([M+H]⁺): 608.2, Found: 608.1;

EXAMPLE.(S)-4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanoicacid (QCO2024). 224 mg of QCO2023 was treated with TFA/DCM (15 mL, 1/3)at 23° C. The reaction was stirred at 23° C. and monitored by TLC. After1.5 hours, starting material was not observed by TLC.

The volatile material was removed under reduced pressure resulting in asemi-solid residue, which was treated with cold Et₂O, to provide a palewhite solid precipitate, which was collected by filtration and dried inair to provide(S)-4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanoicacid QCO2024 (169 mg, 83% for 2 steps). λ_(max)=280 nm; LC-MS (AgilentG6130B Quadrupole LC/MS): Mobile phase: Buffer (pH 7)-CH3CN; Column:Analytic C18 column; Method: 0-100 CH3CN-15 min, t_(R)=3.40 min. MS m/z:MS-API: Calcd. for C₂₂H₂₁F₃N₇O₇ ([M+H]⁺): 552.1, Found: 552.1; ¹H NMR(400 MHz, DMSO) δ=12.16 (s, br, 1H), 8.88 (d, J=7.2 Hz, 1H), 8.65 (s,1H), 7.92 (d, J=8.0 Hz, 2H), 7.64 (d, J=8.0 Hz, 2 H), 7.16 (s, br, 1H),5.14 (s, 2H), 4.38-4.55 (m, 1H), 3.64 (s, 3H), 2.28-2.40 (m, 2H),2.00-2.12 (m, 1H), 1.87-2.00 (m, 1H); ¹³C NMR (101 MHz, DMSO) δ=173.91,172.36, 165.93, 161.03, 156.11, 155.76 (d, J=35.8 Hz), 154.19, 149.40,144.45, 141.80, 134.30, 128.89, 128.62, 128.29, 117.91 (d, J=48.5 Hz),53.90, 52.23, 52.06, 30.26, 25.81; ¹⁹F NMR (377 MHz, CDCl₃) δ=−62.87.

EXAMPLE. Pte-γGlu-Lys-OH (EC1777). EC1777 was prepared using solid phasepeptide synthesis as follows.

Molecular Quantity Compound mmol Equivalent Weight (grams)Fmoc-Lys-Resin 0.5 1 1.00 (Loading ~0.5 mmol/g) Fmoc-Glu-O^(t)Bu 1.0 2425.5 0.426 N¹⁰-TFA-Pteroic 0.65 1.3 408 0.265 Acid PyBOP 1.3 2 520.310.52 DIPEA 1.5 3 129.24 0.168 (d = 0.742)

In a peptide synthesis vessel, Fmoc-Lys-resin (1.0 g, 0.5 mmol) wasplaced and washed with DMF (3×10 ml). Initial Fmoc deprotection wasperformed using 20% piperidine in DMF (3×10 ml) solution for 10 mins percycle. Subsequent washes of DMF (3×10 ml) and i-PrOH (3×10 ml), a Kaisertest was done to determine reaction completion. Following another DMFwash (3×10 ml); an amino acid solution (2.0 eq.) in DMF, PyBOP (2.0 eq.)and DIPEA (3.0 eq.) were added to the vessel and the solution bubbledwith Argon for 1 hour. The coupling solution was filtered, the resin waswashed with DMF (3×10 ml) and i-PrOH (3×10 ml) and a Kaiser test wasdone to assess reaction completion. The above process was performedsuccessively for the additional coupling. Resin cleavage was performedwith a cocktail consisting of 95% CF3CO2H, 2.5% H2O and 2.5%triisopropylsilane. The cleavage cocktail (10 ml) was poured onto theresin and bubbled with Argon for 30 mins, followed by filtration into aclean flask. Further cleavage was performed twice successively withfresh cleavage cocktail for 10 mins of bubbling. The combined filtratewas poured onto cold diethyl ether, the precipitate formed was collectedby centrifugation at 4000 rpm for 5 mins (3×). The precipitate wasobtained following decanting and drying of the solid under vacuum.Deprotection of the trifluoro-acetyl group was achieved by dissolvingthe crude precipitate in H2O (15 ml), which was basified with Na2CO3 topH 9 with Argon bubbling. Upon completion of the reaction, confirmed byLCMS, the solution was acidified to pH 3 using 2 M HCl and the desiredlinker was purified by preparative HPLC (mobile phase A=10 mM Ammoniumacetate, pH=5; Organic phase B=Acetonitrile; Method; 10% B to 100% B in30 mins) to yield EC 1777 (112 mg, 39%); 1H NMR (500 MHz DMSO-d6)Pivotal signals: δ 8.60 (s, 1H), 7.58 (d, 2H), 6.60 (d, 2H), 4.45 (s,2H). [M+H]+=Calculated 570.23, found 570.582

EXAMPLE. Pte-γGlu-Lys-NOTA. In a dry flask, EC 1777 (30.5 mg, 0.054mmol, 1.0 eq.), 1,1,3,3-tetramethylguanidine (13.45 μl, 0.107 mmol, 2.0eq.) and DMSO (2.5 ml) under Argon were sonicated for 1 hour. DIPEA(0.19 ml, 1.07 mmol, 20 eq.) was added to the solution, followed bysonication for an addition hour. To the transparent solution was addedp-SCN-Bn-NOTA.3HCl (33 mg, 0.059 mmol, 1.1 eq.) and the reaction wasmonitored until completion by LCMS and purified using preparative HPLC(mobile phase A=10 mM Ammonium acetate, pH=5; Organic phaseB=Acetonitrile; Method; 10% B to 100% B in 30 mins) to yield EC 1778 (16mg, 29%). 1H NMR (500 MHz DMSO-d6) Pivotal signals: δ 8.60 (s, 1H), 7.58(d, 2H), 7.29 (d, 2H), 7.07 (d, 2H), 6.61 (d,2H), 4.45 (s, 2H), 4.20 (t,1H). [M+H]+=Calculated 1020.39, found 1020.63.

EXAMPLE. Pte-γGlu-Lys-NOTA -Al-18F is prepared by reaction ofPte-γGlu-Lys-NOTA with Al¹⁸F3.3H₂O (1 step method) or with AlCl₃.3H₂Ofollowed by reaction with Na¹⁸F (2 step method) using publishedprocesses.

EXAMPLE. N10-TFA-Pte-γGlu-OtBu-Arg(Pbf)-Arg(Pbf)-Lys(Mtt)-resin 3. Thegeneral procedure described for the synthesis of resin boundfolate-peptide resin 1 was followed for the coupling of 2×Fmoc-L-Arg(Pbf)-OH, Fmoc-Glu-OtBu, and N10-TFA-Pte-OH toFmoc-L-Lys(Mtt)-Wang resin.

EXAMPLE. Pte-γGlu-Arg-Arg-Lys-Bn-NOTA 4 (EC2217). In a peptide synthesisvessel, N10-TFA-Pte-γGlu-OtBu-Arg(Pbf)-Arg(Pbf)-Lys(Mtt)-resin (0.28 g,0.07 mmol) was placed and washed with DCM (3×10 ml). Selective Mttdeprotection was performed by adding a 2% CF₃CO₂H/DCM solution to thevessel and bubbling with Argon for 10 min. After filtering, the resinwas washed with dichloromethane followed by a fresh solution of 2%CF₃CO₂H/DCM. This process was repeated until there was no more yellowsolution being yielded and a Kaiser test was done. Following a DMF wash(3×10 ml); p-SCN-Bn-NOTA.3HCl (50 mg, 0.09 mmol, 1.2 eq.) in DMF, andDIPEA (80 μl, 0.45 mmol, 6.0 eq.) were added to the vessel and thesolution bubbled with Argon for 2 hour. The coupling solution wasfiltered, the resin was washed with DMF (3×10 ml) and i-PrOH (3×10 ml)and a Kaiser test was done to assess reaction completion. Resincleavage/global tert-butyl ester deprotection was performed with acocktail consisting of 95% CF₃CO₂H, 2.5% H₂O and 2.5%triisopropylsilane. The cleavage cocktail (10 ml) was poured onto theresin and bubbled with Argon for 60 mins, followed by filtration into aclean flask. Further cleavage was performed twice successively withfresh cleavage cocktail for 20 mins of bubbling. The combined filtratewas poured onto cold diethyl ether, the precipitate formed was collectedby centrifugation at 4000 rpm for 5 mins (3×). The precipitate wasobtained following decanting and drying of the solid under vacuum.Deprotection of the trifluoro-acetyl group was achieved by dissolvingthe crude precipitate in H₂O (15 ml), which was basified with Na₂CO₃ topH 9 with Argon bubbling. Upon completion of the reaction, confirmed byLCMS, the solution was acidified to pH 5 using 2 M HCl and the desiredlinker was purified by preparative HPLC (mobile phase A=10 mM Ammoniumacetate, pH=5; Organic phase B=Acetonitrile; Method; 10% B to 100% B in30 mins) to yield EC2217 (35 mg, 35%). 1H NMR (500 MHz DMSO-d6) Pivotalsignals: δ 8.61 (s, 1H), 7.54 (d, J=8.4 Hz, 2H), 7.17-7.03 (m, 2H), 6.99(d, J=8.0 Hz, 2H), 6.66 (d, J=8.5 Hz, 2H), 4.52-4.45 (m, 1H), 4.17 (dt,J=8.9, 4.6 Hz, 2H), 4.12 (s, 1H), 4.07-3.97 (m, 1H). [M+H]+=Calculated1332.59, found 1332.87

EXAMPLE.N10-TFA-Pte-γGlu-OtBu-Asp(OtBu)-Arg(Pbf)-Arg(Pbf)-Lys(Mtt)-resin 5. Thegeneral procedure described for the synthesis of resin boundfolate-peptide resin 1 was followed for the coupling of 2×Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-Glu-OtBu, andN10-TFA-Pte-OH to Fmoc-L-Lys(Mtt)-Wang resin.

EXAMPLE. Pte-γGlu-Asp-Arg-Arg-Lys-Bn-NOTA 6 (EC2218).Pte-γGlu-Asp-Arg-Arg-Lys-Bn-NOTA, EC2218 was prepared in 18% yieldaccording to the process described for folate-peptide-NOTA, 4. 1H NMR(500 MHz DMSO-d6) Pivotal signals: δ 8.58 (s, 1H), 7.52 (d, J=9.0 Hz,2H), 7.14-7.08 (m, 4H), 6.61 (d, J=9.0 Hz, 2H), 4.16-4.09 (m, 2H), 4.06(dd, J=10.0, 4.3 Hz, 1H), 3.90 (dd, J=7.8, 4.7 Hz, 1H).[M+H]+=Calculated 1449.64, found 1449.76

EXAMPLE. N10-TFA-Pte-γGlu-OtBu-Arg(Pbf)-Lys(Mtt)-resin 7. The generalprocedure described for the synthesis of resin bound folate-peptideresin 1 was followed for the coupling of Fmoc-L-Arg(Pbf)-OH,Fmoc-Glu-OtBu, and N10-TFA-Pte-OH to Fmoc-L-Lys(Mtt)-Wang resin.

EXAMPLE. Pte-γGlu-Arg-Lys-Bn-NOTA 8 (EC2219). Pte-γGlu-Arg-Lys-Bn-NOTA,EC2219 was prepared in 20% yield according to the process described forfolate-peptide-NOTA, 4. 1H NMR (500 MHz DMSO-d6) Pivotal signals: δ 8.68(s, 1H), 7.60 (d, J=8.4 Hz, 3H), 7.27-6.97 (m, 4H), 6.77-6.69 (m, 2H),4.28-f 4.19 (m, 2H), 4.08 (dd, J=9.0, 5.4 Hz, 1H), 4.01 (dd, J=8.5, 5.4Hz, 1H). [M+H]+=Calculated 1178.51, found 1178.7

EXAMPLE. Pte-γGlu-Arg-Arg-Lys-NOTA 9 (EC2222). In a peptide synthesisvessel, N10-TFA-Pte-γGlu-OtBu-Arg(Pbf)-Arg(Pbf)-Lys(Mtt)-resin (0.5 g,0.12 mmol) was placed and washed with DCM (3×10 ml). Selective Mttdeprotection was performed by adding a 2% CF₃CO₂H/DCM solution to thevessel and bubbling with Argon for 10 min. After filtering, the resinwas washed with dichloromethane followed by a fresh solution of 2%CF₃CO₂H/DCM. This process was repeated until there was no more yellowsolution being yielded and a Kaiser test was done. Following a DMF wash(3×10 ml); NOTA-Bis(tBu)ester (0.10 g, 0.24 mmol, 2.0 eq.) in DMF, PyBOP(0.14 g, 0.26 mmol, 2.2 eq) and DIPEA (64 μl, 0.36 mmol, 3.0 eq.) wereadded to the vessel and the solution bubbled with Argon for 2 hour. Thecoupling solution was filtered, the resin was washed with DMF (3×10 ml)and i-PrOH (3×10 ml) and a Kaiser test was done to assess reactioncompletion. Resin cleavage/global tert-butyl ester deprotection wasperformed with a cocktail consisting of 95% CF₃CO₂H, 2.5% H₂O and 2.5%triisopropylsilane. The cleavage cocktail (10 ml) was poured onto theresin and bubbled with Argon for lhr, followed by filtration into aclean flask. Further cleavage was performed twice successively withfresh cleavage cocktail for 10 mins of bubbling. The combined filtratewas poured onto cold diethyl ether, the precipitate formed was collectedby centrifugation at 4000 rpm for 5 mins (3×). The precipitate wasobtained following decanting and drying of the solid under vacuum.Deprotection of the trifluoro-acetyl group was achieved by dissolvingthe crude precipitate in H₂O (15 ml), which was basified with Na₂CO₃ topH 9 with Argon bubbling. Upon completion of the reaction, confirmed byLCMS, the solution was acidified to pH 5 using 2 M HCl and the desiredlinker was purified by preparative HPLC (mobile phase A=10 mM Ammoniumacetate, pH=5; Organic phase B=Acetonitrile; Method; 10% B to 100% B in30 mins) to yield EC2222 (28mg, 20%). 1H NMR (500 MHz DMSO-d6) Pivotalsignals: δ 8.60 (s, 1H), 7.51 (d, J=8.1 Hz, 2H), 6.64 (d, J=8.4 Hz, 2H),4.21-4.09 (m, 2H), 4.09-4.03 (m, 1H), 3.98-3.88 (m, 1H), 3.50 (s, 1H).[M+H]+=Calculated 1167.57, found 1167.8

EXAMPLE. (S)-Methyl18-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazanonadecan-19-oate(QC07010). QCO2024 (100 mg, 0.181 mmol) is added to a solution ofMono-Boc-PEG-NH₂ (45 mg, 0.181 mmol) and DIPEA (0.158 mL, 0.905 mmol) inDMSO (2 mL) at 23° C. under N₂. After being stirred for 15 min at 23°C., PyBOP (94.2 mg, 0.181 mmol) was added, and the reaction mixture wasstirred for 24 h at 23° C. Volatile material was removed under reducedvacuum, the crude material was further purified by SPE purification:extract successively with ACN (2×), EA (1×) and Et₂O (1×) to afford pureproduct QC07010 (127 mg, 90%). λmax=280 nm; LC-MS (Agilent G6130BQuadrupole LC/MS): Mobile phase: Buffer (pH 7)-CH3CN; Column: AnalyticC18 column; Method: 0-100 CH3CN-15 min, tR=5.06 min. MS m/z: MS-API:Calcd. for C33H43F3N9O10 ([M+H]+): 782.3, Found: 782.2; 1H NMR (400 MHz,DMSO) δ=11.59 (s, br, 1H), 8.92 (d, J=7.2 Hz, 1H), 8.64 (s, 1H),7.85-8.02 (m, 3H), 7.64 (d, J=8.0 Hz, 2H), 6.75 (t, J=5.2 Hz, 1H), 5.13(s, 2H), 4.33-4.48 (m, 1H), 3.64 (s, 3H), 3.46 (s, 4H), 3.30-3.41 (s,4H), 3.14-3.23 (m, 2H), 3.01-3.08 (m, 2H), 2.19-2.30 (m, 2H), 2.02-2.12(m, 1H), 1.89-2.00 (m, 1H), 1.35 (s, 9H); 13C NMR (101 MHz, DMSO)δ=172.43, 171.46, 165.73, 160.87, 156.80, 155.70 (d, J=35.5 Hz), 155.67,154.17, 149.49, 144.20, 141.73, 134.30, 128.82, 128.55, 128.23, 116.20(d, J=290.0 Hz), 77.65, 69.58, 69.50, 69.193, 69.192, 53.88, 52.52,51.96, 38.89, 38.62, 31.65, 28.23, 26.32; 19F NMR (377 MHz, CDCl₃)δ=−62.87.

EXAMPLE. (S)-methyl2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-5-oxopentanoate(QC07011). QC07010 (274 mg, 0.35 mmol) was treated with TFA/DCM (4 mL,1/3) at 23° C. The reaction was stirred at 23° C. and monitored byLC-MS. After 1.5 h, TLC showed that all starting material disappeared.The mixture was diluted with CH3CN and evaporated to dry via rota-yap.Residue TFA (b.p. 72.4° C.) was removed through azeotropic distillationwith ACN to afford the product QC07011 in quantitative yield, which wasused without further purification. λ_(max)=280 nm; LC-MS (Agilent G6130BQuadrupole LC/MS): Mobile phase: Buffer (pH 7)-ACN; Column: Analytic C18column; Method: 0-100 ACN 15 min, t_(R)=3.84 min. MS m/z: MS-API: Calcd.for C₂₈H₃₅F₃N₉O₈ ([M+H]⁺): 682.2, Found: 682.2.

EXAMPLE.(S)-2,2′-(7-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-3,7,18-trioxo-2,11,14-trioxa-8,17-diazanonadecan-19-yl)-1,4,7-triazonane-1,4-diyl)diaceticacid (QC07013). QC07011 (15.7 mg, 0.023 mmol) in DMSO (0.5 ml) was addedNOTA-NHS (18.2 mg, 0.028 mmol) followed by DIPEA (15 μL, 0.084 mmol).The reaction was stirred at 23° C., monitored by LC-MS, and most of thestarting material was converted to QC07013 in 5 hours. The product waspurified by RP-C₁₈ HPLC to afford the pure product QC07013 (13.0 mg,58.5%). λ_(max)=280 nm; LC-MS (Agilent G6130B Quadrupole LC/MS): Mobilephase: Buffer (pH 7)-CH3CN; Method: 0-100 CH3CN-15 min, t_(R)=3.74 min.MS m/z: MS-API: Calcd. for C₄₀H₅₄F₃N₁₂O₁₃([M+H]⁺): 967.4, Found: 967.2;HPLC (Agilent Preparative C18 Column): Mobile phase: Buffer (pH7)-CH3CN; Method: 0-100 CH3CN-30 min, t_(R)=10.75 min

EXAMPLE.(S)-2,2′-(7-(1-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)phenyl)-3-carboxy-1,6,17-trioxo-10,13-dioxa-2,7,16-triazaoctadecan-18-yl)-1,4,7-triazonane-1,4-diyl)diaceticacid (FA-PEG1-NOTA, QC07017). QC07013 (20.8 mg, 0.022 mmol) was stirredin 1.2 mL of 1 M NaOH (aq.) at 23° C. and the reaction was monitored byLC-MS. After 15 min, all starting material was transformed to product,the crude material was purified by RP-C18 HPLC to afford QC07017 (11.3mg, 60%). λmax=280 nm; HPLC (Agilent Preparative C18 Column): Mobilephase: Buffer (pH 7)-CH3CN; Method: 0-30 CH3CN-30 min, tR=11.49 min.LC-MS (Agilent G6130B Quadrupole LC/MS): Mobile phase: Buffer (pH7)-CH3CN; Method: 0-100 CH3CN 15 min, tR=2.72 min. MS m/z: MS-API:Calcd. for C37H53N12O12 ([M+H]+): 857.4, Found: 857.2. 1H NMR (400 MHz,DMSO) δ=8.62 (s, 1H), 8.28 (t, J=5.6 Hz, 1H), 7.99 (t, J=5.6 Hz, 1H),7.85 (d, J=7.2 Hz, 1H), 7.76-7.80 (s, br, 2H), 7.58 (d, J=8.8 Hz, 2H),7.00 (t, J=6.0 Hz, 1H), 6.62 (d, J=8.8 Hz, 2H), 4.47 (d, J=5.2 Hz, 2H),4.13-4.18 (m, 1H), 3.43 (s, 4H), 3.31-3.41 (m, 4H), 3.29-3.32 (m, 2H),3.10-3.24 (m, 4H), 3.03-3.10 (s, br, 2H), 2.90-3.03 (s, br, 2H),2.10-2.14 (m, 2H), 1.97-2.05 (m, 1H), 1.84-1.91 (m, 1H); 13C NMR (101MHz, DMSO) δ=174.33, 172.21, 171.17, 170.35, 165.70, 161.85, 156.19,154.95, 150.56, 148.45, 148.32, 128.62, 127.87, 121.84, 111.38, 69.44,69.30, 69.08, 68.70, 60.95, 57.48, 53.11, 50.85, 49.41, 48.91, 45.88,38.60, 38.18, 32.04, 27.52.

EXAMPLE. Solid Phase Synthesis (SPS) of FA-PEG₆-EDA-NH₂ Precursor(QC03019). 1,2-Diaminoethane trityl resin (1.2 mmol/g, 100 mg, 0.12mmol) was swollen with dichloromethane (DCM, 3mL) followed by dimethylformamide (DMF, 3 mL). After swelling the resin in DMF, a solution offluorenylmethoxycarbonyl (Fmoc)-PEG₆-OH (1.5 equiv), HATU (1.5 equiv),and DIPEA (2.0 equiv) in DMF was added. Argon was bubbled for 2 h, andresin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). The abovesequence was repeated for two more coupling steps for conjugation ofFmoc-Glu-(OtBu)-OH and N¹⁰-TFA-Ptc-OH. The final product was cleavedfrom the resin using a trifluoroacetic acid (TFA):H₂O:triisopropylsilanecocktail (95:2.5:2.5) and concentrated under vacuum. The concentratedproduct was precipitated in diethyl ether and dried under vacuum, whichwas then incubated in Sat. Na₂CO₃ and monitored by LC-MS. 1 hour later,the mixture was neutralized to pH=7 with 2 M HCl (aq.) which waspurified by preparative with preparative RP-C₁₈ HPLC [solvent gradient:0% B to 50% B in 30 min; A=10 mM NH₄OAc, pH=7; B=CH₃CN]. Acetonitrilewas removed under vacuum, and the residue was freeze-dried to yieldQC03019 as a yellow solid (59 mg, 60%). Analytical RP-C₁₈ HPLC:t_(R)=4.22 min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% Bto 50% B in 15 min); Preparative RP-C₁₈ HPLC: t_(R)=11.7 min (A=10 mMNH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% B to 50% B in 30 min);λ_(max)=280 nm; HPLC (Agilent Preparative C18 Column): Mobile phase:Buffer (pH 7)-CH₃CN; Method: 0-30 CH₃CN-30 min, t_(R)=11.7 min LC-MS(Agilent G6130B Quadrupole LC/MS): Mobile phase: Buffer (pH 7)-CH₃CN;Method: 0-50 CH₃CN-15 min, t_(R)=4.22 min MS m/z: MS-API: Calcd. forC₃₆H₅₅N₁₀O₁₂ ([M+H]⁺): 819.4, found, 819.2. ¹H NMR (DMSO-d₆/D₂O) δ=8.63(s, 1H), 7.64 (d, J=8.8 Hz, 2H), 6.64 (d, J=8.8 Hz, 2H), 4.48 (s, 2H),4.12-4.21 (m, 1H), 3.58 (t, J=6.4 Hz, 2H), 3.41-3.53 (m, 24H), 3.18-3.25(m, 2H), 3.11-3.18 (m, 2H), 2.28 (t, J=6.4, 2H), 2.15 (t, J=7.4, 2H),2.03 (m, 1H), 1.88 (m, 1H) ppm.

EXAMPLE. FA-PEG₆-NOTA. To QC03019 (9.5 mg, 0.011 mmol) in DMSO (0.40 ml,with a concentration at 0.029 M) was added NOTA-NHS (8.6 mg, 0.013 mmol)followed by DIPEA (7.0 μL, 0.039 mmol). The reaction was stirred at 23°C., monitored by LC-MS, and most of the starting material wastransformed to the corresponding product in 5 hours. The crude materialwas purified by RP-C₁₈ HPLC to afford the pure product QC07029 (5.5 mg,45%). Analytical RP-C₁₈ HPLC: t_(R)=3.91 min (A=10 mM NH₄OAc, pH=7.0;B=CH₃CN, solvent gradient: 0% B to 50% B in 15 min); Preparative RP-C₁₈HPLC: t_(R)=10.51 min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solventgradient: 0% B to 50% B in 30 min); λ_(max)=280 nm; HPLC (AgilentPreparative C18 Column): Mobile phase: Buffer (pH 7)-CH3CN; Method: 0-30CH₃CN-30 min, t_(R)=10.51 min. LC-MS (Agilent G6130B Quadrupole LC/MS):Mobile phase: Buffer (pH 7)-ACN; Method: 0-50 ACN-15 min, t_(R)=3.91 minMS m/z: MS-API: Calcd. for C₄₈H₇₄N₁₃O₁₇ ([M+H]⁺): 1104.5, Found: 1104.4

EXAMPLE. FA-NOTA-Al—¹⁸F Radiotracer[2]. Two methods for the formation ofFANOTA-Al—¹⁸F are described herein. Conditions including the pH value,concentration of the substrates and temperature for the chelatingreaction with ¹⁸F—Al can be varied. The general methods forFA-NOTA-Al—¹⁸F are described as followed:

Method a). FA-NOTA Precursor was dissolved in 2 mM NaOAc (pH 4.5) and0.5 mL of ethanol, which was treated with Al¹⁸F₃.3H₂O (1.5 eq.) whichwas freshly prepared before application. The pH was adjusted to 4.5-5.0,and the reaction mixture was refluxed for 15-30 min with pH kept at4.5-5.0. After being cooled down to room temperature, the crude materialwas loaded to a cartridge, and the radiotracer was eluted into vial.After sterile filtration and being diluted to appropriate radioactivity(5-10 mCi) and specific activity (>1 Ci/μmol), the radiotracer was readyfor in vivo PET imaging study.

Method b). FA-NOTA Precursor was dissolved in 2 mM NaOAc (pH 4.5), andtreated with AlCl₃.3H₂O (1.5 eq.). The pH was adjusted to 4.5-5.0, andthe reaction mixture was refluxed for 15-30 min with pH kept at 4.5-5.0.The crude material was purified by RP-HPLC to afford the FA-NOTA-Al—OHintermediate ready for ¹⁸F-labeling. Appropriate amount of FA-NOTA-Al—OHwas treated with Na¹⁸F saline solution and ethanol (1/1, v/v), and thewhole mixture was heated at 100-110° C. for 15 min. After being cooleddown to room temperature, the crude material was loaded to a cartridge,and the radiotracer was eluted into vial. After sterile filtration andbeing diluted to appropriate radioactivity (5-10 mCi) and specificactivity (>1 Ci/μmol), the radiotracer was ready for in vivo PET imagingstudy.

EXAMPLE. Standard Protocol for the Formulation of Folate-NOTA-Al¹⁸FRadiotracer. The resin containing ¹⁸F was first washed with 1.5 mL ofultrapure water, and then ¹⁸F was eluted out from resin by using 1.0 mLof 0.4 M KHCO₃ solution. 100 μL of the eluting solution containing ¹⁸Fwas added to a stem vial charged with 10 μL acetic acid, 25 μL AlCl₃ (2mM in 0.1 M NaOAc pH 4 buffer) and 125 μL 0.1 M NaOAc pH 4 buffer. Thewhole mixture was incubated for 2 min before 0.25 mg folate-NOTAprecursor (1) in 125 μL of 0.1 M NaOAc pH 4 buffer was transferred tothe same stem vial. The reaction was immediately heated to 100° C. for15 min.

After cooling to room temperature, the crude material was mixed with 0.7mL 0.1% formic acid and purified by radioactive HPLC on a Xselect CSHC18 (250×10 mm) column using MeCN and 0.1% formic acid as the mobilephase. The fraction at 11.5 min was collected to afford pure radiotracerin ˜40-50% radiochemical yield (RCY) with ˜98% radiochemical purity(RCP). The total radiochemical synthesis of folate-NOTA-Al¹⁸F (2,Al¹⁸F-QC07017) was accomplished in ˜37 min with a specific activity (SA)of 70±18.4 GBq/μmol. After sterile filtration and appropriate dilutionin isotonic saline to the desired radioactivity, the folate-NOTA-Al¹⁸F(2) radiotracer was ready for PET imaging study.

Using same strategy, radiochemcial synthesis of FA-PEG₁₂-NOTA-Al—¹⁸Fradiotracer (QC07043) was accomplished in ˜35 min with a specificactivity (SA) of 49±17.1 GBq/μmol. Although the radiochemical purity isexcellent, 100% after radioactive HPLC purification, the totalradiochemical yield (RCY) is relatively low, ˜25-30%. After sterilefiltration and appropriate dilution in isotonic saline to the desiredradioactivity, the FA-PEG₁₂-NOTA-Al—¹⁸F radiotracer was ready for PETimaging study.

EXAMPLE. Solid Phase Synthesis (SPS) of FA-PEG₁₂-EDA-NH₂ (QC07042)[11].1,2-Diaminoethane trityl resin (1.2 mmol/g, 50 mg, 0.06 mmol) wasswollen with dichloromethane (DCM, 3mL) followed by dimethyl formamide(DMF, 3 mL). After swelling the resin in DMF, a solution offluorenylmethoxycarbonyl (Fmoc)-PEG₁₂-OH (1.5 equiv), HATU (1.5 equiv),and DIPEA (2.0 equiv) in DMF was added. Argon was bubbled for 2 h, andresin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). The abovesequence was repeated for two more coupling steps for conjugation ofFmoc-Glu-(OtBu)-OH and N¹⁰-TFA-Ptc-OH. The final product was cleavedfrom the resin using a trifluoroacetic acid (TFA):H₂O:triisopropylsilanecocktail (95:2.5:2.5) and concentrated under vacuum. The concentratedproduct was precipitated in diethyl ether and dried under vacuum, whichwas then incubated in Sat. Na₂CO₃ and monitored by LC-MS. 1 hour later,the mixture was neutralized to pH=7 with 2 M HCl (aq.) which waspurified by preparative with preparative RP-C₁₈ HPLC [solvent gradient:0% B to 50% B in 30 min; A=10 mM NH₄OAc, pH=7; B=CH₃CN]. Acetonitrilewas removed under vacuum, and the residue was freeze-dried to yield pureQC07042 as a yellow solid (32.5 mg, 50%). Analytical RP-C₁₈ HPLC:t_(R)=4.76 min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% Bto 50% B in 15 min); Preparative RP-C₁₈ HPLC: t_(R)=13.75 min (A=10 mMNH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% B to 50% B in 30 min);UV-Vis: λ_(max)=280 nm; Preparative RP-C₁₈ HPLC: HPLC (AgilentPreparative C18 Column): Mobile phase: Buffer (pH 7)-CH₃CN; Method: 0-50CH₃CN, 30 min, t_(R)=13.75 min. LC-MS: LC-MS (Agilent G6130B QuadrupoleLC/MS) of Product Mobile phase: Buffer (pH 7)-CH₃CN; Method: 0-50 CH₃CN,15 min, t_(R)=4.76 min. MS m/z: MS-API: Calcd. for C₄₈H₇₉N₁₀O₁₈([M+H]⁺): 1083.6, Found: 1083.4;

EXAMPLE. FA-PEG12-EDA-NH₂-NOTA (QC07043). To FA-PEG12-EDA-NH₂ (QC07042,4.78 mg, 0.004 mmol, M.W.: 1082.5) in DMSO (0.25 ml, with aconcentration at 0.025 M) was added NOTA-NHS (3.5 mg, 0.005 mmol, 1.2eq.) followed by DIPEA (2.7 μL, 0.039 mmol). The whole mixture wasstirred at 23° C. and monitored by LC-MS. 4 hours later, LC-MS showedthat almost all of the starting material was transformed to the product.The crude material was then purified by preparative RP-HPLC to affordthe pure FA-PEG12-EDA-NH₂-NOTA (QC07043, 4.09 mg, 68%). AnalyticalRP-C₁₈ HPLC: t_(R)=6.21 min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solventgradient: 0% B to 30% B in 15 min); Preparative RP-C₁₈ HPLC: t_(R)=15.60min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% B to 30% B in30 min); UV-Vis: λ_(max)=280 nm; LC-MS: LC-MS (Agilent G6130B QuadrupoleLC/MS) of Product Mobile phase: Buffer (pH 7)-CH₃CN; Method: 0-8.00 (m,1H), 7.55 (d, J=6.4 Hz, 1H), 7.54 (s, br, 2H), 6.81-6.93 (m, 1H), 6.62(d, J=8.0 Hz, 2H), 4.45 (d, J=4.4 Hz, 2H), 3.95-4.03 (m, 1H), 3.64-3.70(m, 2H), 3.56-3.63 (m, 6H), 3.38-3.50 (m, 28H), 3.33-3.36 (m, 6H),3.20-3.24 (m, 4H), 3.09-3.18 (m, 10H), 3.04-3.09 (m, 4H), 2.50 (s, 12H,overlapping with the residue peak of DMSO), 2.27-2.34 (m, 2H), 2.02-2.12(m, 2H), 1.99-2.01 (m, 2H).

EXAMPLE. C-NETA and folate-C-NETA. A PyBOP promoted coupling betweenQC04018 and compound 6, followed by deprotection of tert-butyl esterwith TFA, provided folate-C-NETA. The folate-C-NETA is used to evaluatethe labeling efficiency with Al¹⁸F and ⁶⁸Ga and evaluate the in vivo PETimaging.

EXAMPLE. Methyl 3-cyano-4-(dimethylamino)benzoate (QC07002)[1]. To astirred solution of methyl 3-cyano-4-fluorobenzoate (5 g, 27.9 mmol) inDMSO (6 ml) was added dimethylamine hydrochloride (2.75 g, 33.7 mmol)followed by potassium carbonate (8.1 g, 58.6 mmol). The reaction mixturewas stirred at room temperature overnight and concentrated. The residuewas dissolved in dichloromethane (50 ml) and washed with water (2×25ml), brine, dried over Na₂SO₄ and concentrated in vacuo to give themethyl 3-cyano-4-(dimethylamino)benzoate (QC07002) in quantitative yieldand was used without further purification.

EXAMPLE. 2-Cyano-4-(methoxycarbonyl)-N,N,N-trimethylbenzenaminiumtrifluoromethanesulfonate (QC07003). To a stirred solution of methyl3-cyano-4-(dimethylamino)benzoate (3.4 g, 16.7 mmol) in anhydrousdichloromethane (17 ml) was added methyl trifluoromethanesulfonate (10g, 60.9 mmol, M.W. 164.1) dropwise. The reaction was stirred at RT for16 h and another portion of methyl trifluoromethanesulfonate (10 g, 60.9mmol, M.W.: 164.1) was added. The reaction was stirred for another 16hours and tert-butylmethylether (20 ml) was added slowly. The suspensionwas filtered and the collected solid was washed withtert-butylmethylether. The crude product was purified by RP-C₁₈ HPLC:(acetonitrile/water-gradient 1:99 to 80:20) to afford product QC07003(3.69 g) in 60% yield. Analytical RP-C₁₈ HPLC: t_(R)=0.49 min (A=10 mMNH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% B to 100% B in 15 min);λ_(max)=275 nm; LC-MS (Agilent G6130B Quadrupole LC/MS): Mobile phase:Buffer (pH 7)-CH3CN; Column: Analytic C₁₈ column; Method: 0-100 CH₃CN-15min, t_(R)=0.49 min. MS m/z: MS-API: Calcd. for C₁₂H₁₅N₂O₂ ([M]⁺):219.1, Found: 219.0; 1H NMR (400 MHz, D₂O) δ=8.67 (d, J=2.1 Hz, 1H),8.44 (dd, J=9.1, 2.1 Hz, 1H), 8.15 (d, J=9.1 Hz, 1H), 3.93 (s, 3H), 3.87(s, 9H) ppm.

EXAMPLE. 4-Carboxy-2-cyano-N,N,N-trimethylbenzenaminiumtrifluoromethanesulfonate (QC07004). A solution of QC07003 (3.6 g, 9.8mmol) in water (83 ml) and TFA (83 ml) was heated at 120° C. for 48 h.The reaction mixture was concentrated in vacuo, the light green oil wastreated with diethylether to result a suspension. This solid wascollected by filtration, washed with diethylether and dried in vacuo togive 4-carboxy-2-cyano-N,N,Ntrimethylbenzenaminiumtrifluoromethanesulfonate QC07004 (2.8 g, 82%). Analytical RP-C₁₈ HPLC:t_(R)=0.61 min (A=10 mM NH4OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% Bto 100% B in 15 min); λ_(max)=240 nm. LC-MS (Agilent G6130B QuadrupoleLC/MS): Mobile phase: Buffer (pH 7)-CH₃CN; Column: Analytic C₁₈ column;Method: 0-100 CH₃CN 15 mM, t_(R)=0.61 min. MS m/z: MS-API: Calcd. forC₁₁H₁₃N₂O₂([M]⁺): 205.1, Found: 205.1; 1H NMR (400 MHz, DMSO) δ=8.58 (d,J=2.07 Hz, 1H), 8.39-8.49 (m, 1H), 8.23-8.35 (m, 1H), 3.85 (s, 9H).

EXAMPLE. FA-PEG₁-TMA Precursor (QC07005). QC07004 (62 mg, 0.17 mmol) wasadded to the solution of QC07011 (0.14 mmol) and DIPEA (87 μL, 1.75mmol) in DMSO (2.0 mL) at 23° C. under N₂. After being stirred for 15min at 23° C., PyBOP (91 mg, 0.17 mmol) was added, and the reactionmixture was stirred for 24 h at 23° C. Volatile material was removedunder reduced vacuum to afford the crude product which was furtherpurified by RP-HPLC (C₁₈) to afford the pure compound QC07005 as paleyellow colored solid (125.1 mg, 72%). Analytical RP-C₁₈ HPLC: t_(R)=4.17min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% B to 100% Bin 15 min); λ_(max)=280 nm; LC-MS (Agilent G6130B Quadrupole LC/MS):Mobile phase: Buffer (pH 7)-CH₃CN; Column: Analytic C₁₈ column; Method:0-100 CH₃CN-15 min, t_(R)=4.17 min. MS m/z: MS-API: Calcd. forC₂₈H₃₅F₃N₉O₈ ([M]⁺): 868.3, Found: 868.2.

EXAMPLE. General procedure for the one-pot ¹⁹F-introduction anddeprotection. 8.3 μL of freshly prepared KF-Kryptofix (1/1.5) (0.0012mmol, 0.144 M) solution was azeotropically dried with CH₃CN at 90-100°C., to which 1.2 mg (0.0012 mmol, 1.0 equiv.) QC07005 in 50 ul ofanhydrous DMSO was added with a concentration of precursor at 0.024 M.The resulting mixture was immediately immersed into an oil bathpreheated to 70-75° C. and kept at 70-75° C. for 10 min. After beingcooled down to room temperature, 200 ul of 1M NaOH (aq.) was added witha concentration of NaOH (aq.) at 0.8 M. The reaction was monitored byLC-MS and found complete after 5 min, which was neutralized with 1 M HCl(aq.) and analyzed by LC-MS (QC07006). And the total labeling efficiencywas about 30% based on the analysis of LC-MS. Analytical RP-C₁₈ HPLC:A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% B to 100% B in 15min; λ_(max)=280 nm; LC-MS: Method: 0-100 CH₃CN-15 min, t_(R)=5.13 minMS m/z: MS-API: Calcd. for C₃₆H₃₇F₄N₁₀O₉ ([M+H]⁺): 829.3, Found: 829.1.

EXAMPLE. Folate-¹⁸F-Boronate PET Imaging Agent

PSMA TARGETED EXAMPLES

EXAMPLE. EC1380, 10. In a dry flask, H-Glu(O^(t)Bu)-O^(t)Bu.HCl (2.48 g,8.41 mmol) and 4-nitrophenyl chloroformate (1.86 g, 9.25 mmol, 1.1 eq)were added, dissolved in CH₂Cl₂ (30 ml) under Argon atmosphere. Thestirring solution was chilled to 0° C., followed by the dropwiseaddition of DIPEA (4.50 ml, 25.2 mmol, 3 eq). The reaction mixture wasallowed to warm to room temperature and stirred for 1 hr. To thestiffing solution was added H-Lys-(Z)—O^(t)Bu (4.39 g, 11.8 mmol, 1.4eq), DIPEA (4.50 ml, 25.2 mmol, 3 eq) and stirred for 1 hr. Uponcompletion, the reaction was quenched with saturated NaHCO₃ andextracted with CH₂Cl₂ three times. The organic extracts were combined,dried over Na₂SO₄, filtered and the solvent was removed via reducedpressure. The product was purified using silica gel chromatography withpetroleum ether and ethyl acetate. The Cbz protected amine wastransferred to a round bottom flask with 10% Pd/C (10% wt eq), dissolvedin MeOH (30 ml) under Hydrogen atmosphere (1 atm) and stirred for 3 hr.Upon completion, the reaction mixture was filtered through celite andthe solvent was removed via reduced pressure to yield the crude amine.The amine was taken up in CH₂Cl₂ (30 ml) under Argon atmosphere andchilled to 0° C. To the chilled solution was added 4-nitrophenylchloroformate (2.2 g, 10.9 mmol, 1.3 eq) and DIPEA (6.0 ml, 33.6 mmol, 4eq) subsequently and stirred for 2 hr at room temperature. The reactionmixture was quenched with saturated NH₄Cl and extract three times withethyl acetate. The organic extracts were combined, dried over Na₂SO₄,filtered, and solvent was removed under vacuum and purified using silicagel chromatography to yield the desired activated amine, EC1380 (2.54 g,46%).

EXAMPLE.Glu(O^(t)Bu)—O^(t)Bu-Lys-O^(t)Bu-AMPAA-Asp(O^(t)Bu)-Asp(O^(t)Bu)-Lys(Mtt)-resin11. The general procedure described for the synthesis of resin boundfolate-peptide resin 1 was followed for the coupling of 2×Fmoc-L-Asp(O^(t)Bu)-OH, Fmoc-AMPAA-OH, Fmoc-L-Lys(Z)-O^(t)Bu, andFmoc-(L)-Glu(O^(t)Bu) to Fmoc-L-Lys(Mtt)-Wang resin. The resin boundpenta-peptide was subjected to standard Fmoc deprotection, washings andKaiser test. Following another DMF wash (3×10 ml); an EC1380 solution(2.0 eq.) in DMF, and DIPEA (3.0 eq.) were added to the vessel and thesolution bubbled with Argon for 2 hour. The coupling solution wasfiltered, the resin was washed with DMF (3×10 ml) and i-PrOH (3×10 ml)and a Kaiser test was done to assess reaction completion.

EXAMPLE. Glu-Lys-AMPAA-Asp-Asp-Lys-Bn-NOTA 12.Glu-Lys-AMPAA-Asp-Asp-Lys-Bn-NOTA, EC2209 was prepared in 47% yieldaccording to the process described for folate-peptide-NOTA, 4. ¹H NMR(500 MHz DMSO-d₆) Pivotal signals: δ 7.25-7.18 (m, 2H), 7.14 (d, J=8.1Hz, 1H), 7.12-7.06 (m, 5H), 4.47 (ddd, J=17.8, 7.5, 5.6 Hz, 2H),4.11-4.08 (m, 3H), 4.08-4.02 (m, 2H), 3.98 (dd, J=8.2, 5.1 Hz, 1H).[M+H]⁺=Calculated 1319.50, found 1319.70

EXAMPLE.Glu(O^(t)Bu)-O^(t)Bu-Lys-O^(t)Bu-Aoc-Phe-Phe-Arg(Pbf)-Asp(O^(t)Bu)-Arg(Pbf)-Lys(Mtt)-resin13. The general procedure described for the synthesis of resin boundfolate-peptide resin 1 was followed for the coupling ofFmoc-L-Arg(Pbf)-OH, Fmoc-L-Asp(O^(t)Bu)-OH, Fmoc-L-Arg(Pbf)-OH, 2×Fmoc-Phe-OH, Fmoc-Aoc-OH, Fmoc-L-Lys(Z)-O^(t)Bu, Fmoc-(L)-Glu(O^(t)Bu)and EC1380 to Fmoc-L-Lys(Mtt)-Wang resin.

EXAMPLE. Glu-Lys-Aoc-Phe-Phe-Arg-Asp-Arg-Lys-NOTA 14.Glu-Lys-Aoc-Phe-Phe-Arg-Asp-Arg-Lys-NOTA, EC2390 was prepared in 37%yield according to the process described for folate-peptide-NOTA, 4. ¹HNMR (500 MHz DMSO-d₆) Pivotal signals: δ 7.25-7.14 (m, 6H), 7.16-7.08(m, 3H), 4.47 (dd, J=9.0, 4.7 Hz, 1H), 4.42 (t, J=5.9 Hz, 1H), 4.36 (dd,J=10.4, 4.4 Hz, 1H), 4.27 (t, J=6.9 Hz, 1H), 4.16 (t, J=5.6 Hz, 1H),3.97-3.88 (m, 2H). [M+H]⁺=Calculated 1639.84, found 1640.22

EXAMPLE. DUPA-EAOA-Phe-Arg-Lys-NH₂.2-[3-(3-Benzyloxycarbonyl-1-tert-butoxycarbonyl-propyl)-ureido]pentanedioicacid di-tert-butyl ester (2). [1, 2] To a solution of L-glutamatedi-tert-butyl ester hydrochloride 1 (1.0 g, 3.39 mmol) and triphosgene(329.8 mg, 1.12 mmol) in DCM (25.0 mL) at −78° C., triethylamine (TEA,1.0 mL, 8.19 mmol) was added. After stirring for 2 h at −78° C. underargon, a solution of L-Glu(OBn)-OtBu (1.2 g, 3.72 mmol) and TEA (600 μL,4.91 mmol) in DCM (5.0 mL) was added. The reaction mixture was allowedto come to room temperature (rt) over a period of 1 h and stirred atambient temperature overnight. The reaction was quenched with 1 M HCl,and the organic layer was washed with brine and dried over Na₂SO₄. Thecrude product was purified using flash chromatography (hexane:EtOAc)1:1) to yield the intermediate 2 (1.76 g, 90.2%) as a colorless oil andcrystallized using hexane:DCM. Rf) 0.67 (hexane:EtOAc) 1:1). ¹H NMR(CDCl₃): δ 1.43 (s, 9H, CH3-tBu); 1.44 (s, 9H, CH3-tBu); 1.46 (s, 9H,CH3-tBu); 1.85 (m, 1H, Glu-H); 1.87 (m, 1H, Glu-H); 2.06 (m, 1H, Glu-H);2.07 (m, 1H, Glu-H); 2.30 (m, 2H, Glu-H); 2.44 (m, 2H, Glu-H); 4.34 [s(broad), 1H, RH]; 4.38 [s (broad), 1H, R—H]; 5.10 (s, 2H, CH2-Ar); 5.22[s (broad), 2H, Urea-H); 7.34 (m, 5H, Ar—H). EI-HRMS (m/z): (M+H)+ calcdfor C₃₀H₄₇N₂O₉, 579.3282; found, 579.3289.

EXAMPLE. 2-[3-(1,3-Bis-tert-butoxycarbonyl-propyl)-ureido]pentanedioicAcid 1-tert-Butyl Ester, DUPA_1. To a solution of 2 (250 mg, 432 mmol)in DCM, 10% Pd/C was added. The reaction mixture was hydrogenated at 1atm for 24 h at rt. Pd/C was filtered through a Celite pad and washedwith DCM. The crude product was purified using flash chromatography(hexane: EtOAc) 40:60) to yield DUPA_1 (169 mg, 80.2%) as a colorlessoil, and crystallized using hexane:DCM. R_(f)=0.58 (hexane:EtOAc=40:60). ¹H NMR (CDCl₃): δ 1.46 (m, 27H, CH3-tBu); 1.91 (m,2H,Glu-H); 2.07 (m, 1H, Glu-H); 2.18 (m,1H, Glu-H); 2.33 (m, 2H, Glu-H);2.46 (m, 2H, Glu-H); 4.31(s (broad), 1H, RH); 4.35 (s (broad), 1H, R—H);5.05 (t, 2H,Urea-H); EI-HRMS (m/z): (M+H)⁺ calcd forC₂₃H₄₁N₂O₉,489.2812; found, 489.2808.

Reagents and conditions: (a) (i) 20%piperidine/DMF, room temperature, 10min; (ii) Fmoc-Arg(Boc)2-OH, HBTU, HOBt, DMF-DIPEA, 2 h. (b) (i) 20%piperidine/DMF, room temperature, 10 min; (ii) Fmoc-Phe-OH, HBTU, HOBt,DMF-DIPEA, 2 h. (c) (i) 20% piperidine/DMF, room temperature, 10 min;(ii) Fmoc-8-amino-octanoic(EAO)acid, HBTU, HOBt, DMF/DIPEA, 2 h. (d) (i)20% piperidine/DMF, room temperature, 10 min; (ii) (tBuO)3-DUPA-OH,HBTU, HOBt, DIPEA, 2 h. (e) TFA/H2O/TIPS (95:2.5:2.5), 1 h

EXAMPLE. DUPA-EAOA-Phe-Arg-Lys-NH₂. Fmoc-Lys(Boc)-Wang resin (0.43 mM)was swollen with DCM (3 mL) followed by dimethyl formamide (DMF, 3 mL).A solution of 20% piperidine in DMF (3×3 mL) was added to the resin, andargon was bubbled for 5 min. The resin was washed with DMF (3×3 mL) andisopropyl alcohol (i-PrOH, 3×3 mL). Formation of free amine was assessedby the Kaiser test. After swelling the resin in DMF, a solution ofFmoc-Arg(Boc)₂-OH (2.5 equiv), HBTU (2.5 equiv), HOBt (2.5 equiv), andDIPEA (4.0 equiv) in DMF was added. Argon was bubbled for 2 h, and resinwas washed with DMF (3×3 mL) and i-PrOH (3×3 mL). The couplingefficiency was assessed by the Kaiser Test. The above sequence wasrepeated for 3 more coupling steps to introduce the phenylanaline (Phe),8-amino-octanoic acid (EAO), and DUPA successively. Final compound wascleaved from the resin using a trifluoroacetic acid(TFA):H₂O:triisopropylsilane cocktail (95:2.5:2.5) and concentratedunder vacuum. The concentrated product was precipitated in cold diethylether and dried under vacuum. The crude product was purified usingpreparative RP-HPLC [(λ) 210 nm; solvent gradient: 0% B to 50% B in 30min run; mobile phase: A) 0.1% TFA, pH=2; B) acetonitrile (ACN)]. ACNwas removed under vacuum, and pure fractions were freeze-dried to yieldDUPA-EAOA-Phe-Arg-Lys-NH₂ as a white solid. UV/vis: λ_(max)=205 nm.Analytical RP-HPLC: t_(R)=6.2 min (A=0.1% TFA; B=CH₃CN, solventgradient: 0% B to 50% B in 15 min); ESI-MS (m/z): (M+H)⁺ calcd forC₄₀H₆₅N₁₀O₁₃, 893.5; found, 893.4.

EXAMPLE. DUPA-EAOA-Phe-Arg-Lys-NH₂-NOTA. To DUPA-EAOA-Phe-Arg-Lys-NH₂(QC08001, 5.0 mg, 0.0056 mmol, M.W.: 893.0) in DMSO (0.20 ml, with aconcentration at 0.028 M) was added NOTA-NHS (5.5 mg, 0.0084 mmol, 1.5eq.) followed by DIPEA (2.9 μL, 0.017 mmol). The reaction was stirred at23° C., monitored by LC-MS, and most of the starting material wastransformed to the corresponding product in 5 hours. The crude materialwas purified by RP-C₁₈ HPLC. ACN was removed under vacuum, and purefractions were freeze-dried to yield the pureDUPA-EAOA-Phe-Arg-Lys-NH₂-NOTA (QC08002, 3.3 mg, 50%). Analytical RP-C₁₈HPLC: t_(R)=5.98 min (A=0.1% TFA; B=CH₃CN, solvent gradient: 0% B to 50%B in 15 min); Preparative RP-C₁₈ HPLC: t_(R)=16.16 min (A=0.1% TFA;B=CH₃CN, solvent gradient: 0% B to 50% B in 30 min); UV-Vis: λ_(max)=201nm; HPLC (Agilent Preparative C18 Column): Mobile phase: A=0.1% TFA;B=CH₃CN; Method: 0-50 CH₃CN-30 min, t_(R)=16.16 min LC-MS (AgilentG6130B Quadrupole LC/MS): Mobile phase: A=0.1% TFA; B=CH₃CN; Method:0-50 CH₃CN-30 min, t_(R)=5.98 min; MS m/z: MS-API: Calcd. forC₅₂H₈₄N₁₃O₁₈ ([M+H]⁺): 1178.6, Found: 1178.4.

EXAMPLE. DUPA-EAOA-Phe-Arg-Lys-NH₂-NOTA-Al¹⁸F. Method a):DUPA-EAOA-Phe-Arg-Lys-NH2-NOTA is dissolved in 2 mM NaOAc (pH 4.5) and0.5 mL of ethanol, and treated with Al¹⁸F₃.3H₂O (1.5 eq.) which isfreshly prepared before application. The pH is adjusted to 4.5-5.0, andthe reaction mixture is refluxed for 15-30 min with pH kept at 4.5-5.0.After cooling to room temperature, the crude material is loaded to acartridge, and the radiotracer eluted into vial. After sterilefiltration and being diluted to appropriate radioactivity (5-10 mCi) andspecific activity (>1 Ci/μmol), the radiotracer is used in in vivo PETimaging.

Method b). DUPA-EAOA-Phe-Arg-Lys-NH2-NOTA is dissolved in 2 mM NaOAc (pH4.5), and treated with AlCl₃.3H₂O (1.5 eq.). The pH is adjusted to4.5-5.0, and the reaction mixture is refluxed for 15-30 min with the pHkept at 4.5-5.0. The crude material is purified by RP-HPLC to afford theDUPA-EAOA-Phe-Arg-Lys-NH₂-NOTA-Al—OH intermediate ready for¹⁸F-labeling. Appropriate amount of DUPA-EAOA-Phe-Arg-Lys-NH₂-NOTA-Al—OHis treated with Na¹⁸F saline solution and ethanol (1/1, v/v), and thewhole mixture is heated at 100-110° C. for 15 min. After cooling to roomtemperature, the crude material is loaded to a cartridge, and theradiotracer eluted into vial. After sterile filtration and being dilutedto appropriate radioactivity (5-10 mCi) and specific activity (>1Ci/μmol), the radiotracer is ready for use in in vivo PET imaging.

Reagents and conditions: (a) Fmoc-Phe-OH, HBTU, HOBt, DMF/DIPEA, 2 h.(b) (i) 20% piperidine/DMF, room temperature, 10min; (ii) Fmoc-Phe-OH,HBTU, HOBt, DMF/DIPEA, 2 h. (c) (i) 20% piperidine/DMF, roomtemperature, 10 min; (ii) Fmoc-8-amino-octanoic (EAO) acid, HBTU, HOBt,DMF/DIPEA, 2 h. (d) (i) 20% piperidine/DMF, room temperature, 10 min;(ii) (tBuO)3-DUPA-OH, HBTU, HOBt, DIPEA, 2 h. (e) TFA/H₂O/TIPS(95:2.5:2.5), 1 h.

EXAMPLE. Solid Phase Peptide Synthesis (SPPS) ofDUPA-EAOA-Phe-Phe-EDA-NH₂.[2, 3]. As described herein forDUPA-EAOA-Phe-Arg-Lys-NH₂ (QC08001), DUPA-EAOA-Phe-Phe-EDA-NH₂ ispreapred. The commercially available Trt-EDA resin was swollen with DCM(3 mL) followed by dimethyl formamide (DMF, 3 mL), to which a solutionof Fmoc-Phe-OH (2.5 equiv), HBTU (2.5 equiv), HOBt (2.5 equiv), andDIPEA (4.0 equiv) in DMF was added. Argon was bubbled for 2 h, and resinwas washed with DMF (3×3 mL) and i-PrOH (3×3 mL). The couplingefficiency was assessed by the Kaiser Test. A solution of 20% piperidinein DMF (3×3 mL) was added to the resin, and argon was bubbled for 5 min.The resin was washed with DMF (3×3 mL) and isopropyl alcohol (i-PrOH,3×3 mL). Formation of free amine was assessed by the Kaiser test. Theabove sequence was repeated for 3 more coupling steps to introduce thesecond phenylanaline (Phe), 8-amino-octanoic acid (EAO), and DUPAsuccessively. Final compound was cleaved from the resin using atrifluoroacetic acid (TFA):H₂O:triisopropylsilane cocktail (95:2.5:2.5)and concentrated under vacuum. The concentrated product was precipitatedin cold diethyl ether and dried under vacuum. The crude product waspurified using preparative RP-HPLC [λ] 210 nm; solvent gradient: 0% B to100% B in 30 min run; mobile phase: A) 10 mM NH₄OAc (pH=7, buffer); B)acetonitrile (ACN)]. ACN was removed under vacuum, and pure fractionswere freeze-dried to yield DUPA-EAOA-Phe-Phe-EDA-NH₂ as a white solid.Analytical RP-C₁₈ HPLC: t_(R)=3.99 min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN,solvent gradient: 0% B to 100% B in 15 min); Preparative RP-C₁₈ HPLC:t_(R)=16.05 min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% Bto 100% B in 30 min); UV-Vis: λ_(max)=209 nm; LC-MS: LC-MS (AgilentG6130B Quadrupole LC/MS) of Product Mobile phase: Buffer (pH 7)-CH₃CN;Method: 0-100 ACN-15 mM, t_(R)=3.99 min. MS m/z: MS-API: Calcd. forC₃₉H₅₆N₇O₁₁ ([M+H]⁺): 798.4, Found: 798.3; Calcd. for C₃₉H₅₅N₇O₁₁K([M+K]⁺): 836.4, Found: 836.3. HPLC (Agilent Preparative C18 Column):Mobile phase: Buffer (pH 7)-CH₃CN; Method: 0-100 ACN-30 min, t_(R)=16.05min.

EXAMPLE. To DUPA-EAOA-Phe-Phe-EDA-NH₂ (QC08008, 5.9 mg, 0.0074 mmol,M.W.: 797.4) in DMSO (0.25 ml, with a concentration at 0.025 M) wasadded NOTA-NHS (7.3 mg, 0.011 mmol, 1.5 eq.) followed by 4 drops ofDIPEA. The mixture was stirred at 23° C. and monitored by LC-MS. 4 hourslater, LC-MS showed that almost all of the starting material wastransformed to the product. The crude material was then purified bypreparative RP-HPLC to afford the pure DUPA-EAOA-Phe-Phe-NOTA (QC08009,4.50 mg, 56%, based on 8.02 mg in theory, 97% purity by HPLC at 210 nm).Analytical RP-C₁₈ HPLC: t_(R)=3.45 min (A=10 mM NH₄OAc, pH=7.0; B=CH₃CN,solvent gradient: 0% B to 100% B in 15 min); Preparative RP-C₁₈ HPLC:t_(R)=10.09 min (A=10 mM NH4OAc, pH=7.0; B=CH₃CN, solvent gradient: 0% Bto 100% B in 30 min); UV-Vis: λ_(max)=211 nm; LC-MS: LC-MS (AgilentG6130B Quadrupole LC/MS) of Product Mobile phase: Buffer (pH 7)-CH₃CN;Method: 0-100 ACN-15 min, t_(R)=3.45 min. MS m/z: MS-API: Calcd. forC₅₁H₇₅N₁₀O₁₆ ([M+H]⁺): 1083.5, Found: 1083.3; HPLC (Agilent PreparativeC18 Column): Mobile phase: Buffer (pH 7)-CH₃CN; Method: 0-100 ACN-30min, t_(R)=10.09 min. ¹H NMR (400 MHz, DMSO-d₆) δ=10.13 (br, 1H), 8.98(br, 1H), 8.43 (br, 1H), 7.90 (br, 3H), 7.30-7.10 (m, 10H), 6.37 (br,1H), 6.28 (br, 1H), 4.60-4.52 (m, 1H), 4.32-4.44 (m, 1H), 4.24-4.31 (m,2H), 3.95-4.03 (m, 2H), 3.85-3.92 (m, 2H), 3.28 (s, 4H), 3.25 (s, 2H),3.09 (m, 1H), 3.05 (m, 1H), 2.92-3.02 (m, 4H), 2.54-2.67 (m, 12H),2.31-2.38 (m, 2H), 2.19-2.31 (m, 3H), 2.11-2.18 (m, 2H),2.02-2.10 (m,3H), 1.52-1.72 (m, 4H), 1.25-1.37 (m, 4H), 1.05-1.13 (m, 2H),

EXAMPLE. Radiochemical Synthesis of DUPA-EAOA-Phe-Arg-Lys-NOTA-⁶⁴CuRadiotracer. NOTA based chelators have also been reported and employedin the formulation of NOTA-^(64/67)Cu for nuclearmedicine/radiotherapy.[14-16] The corresponding DUPA-NOTA-⁶⁴Cu wasprepare for the dual purpose of imaging and therapy, also referred to astheranostics. DUPA-EAOA-Phe-Arg-Lys-NOTA-⁶⁴Cu was prepared according astandard protocol with minor modifications. [4, 14-16] The ⁶⁴Cu(OAc)₂,in situ prepared from ⁶⁴CuCl₂ with 0.1 M ammonium acetate (pH 5.5),wasadded to the reaction tube containing the DUPA-NOTA precursor. Theresulting mixture was then heated to 95° C. for 15 min. After cooling toroom temperature, the crude material was purified by radioactive HPLC ona C18 column using MeCN and 0.1% TFA as the mobile phase to afford thetarget radiotracer with ˜90% radiochemical purity (RCP). Sterilefiltration and dilution in isotonic saline to the desired radioactivityprovided the radiotracer ready for PET imaging.

EXAMPLE. Radiochemical synthesis of DUPA-EAOA-Phe-Phe-NOTA-⁶⁴Cu/Al—¹⁸F.

EXAMPLE. Radiochemical synthesis of DUPA-EAOA-Phe-Phe-NOTA-⁶⁸Ga.

EXAMPLE. General procedure for ⁶⁸Ga labeling: ⁶⁸Ga was eluted from the⁶⁸Ge/⁶⁸Ga generator with 0.1N HCl. A predetermined amount of ⁶⁸Ga in0.1N HCl was added to a DUPA-NOTA solution in acetate buffer (pH 4.8).The labeling mixture was incubated at room temperature, and labelingefficiencies were checked by radioactive HPLC. The radiolabeled productwas purified by radioactive HPLC and the DUPA-NOTA-⁶⁸Ga peak sample wascollected. After sterile filtration and being diluted to appropriateradioactivity (5-10 mCi) and specific activity (>1 Ci/μmol), theradiotracer was ready for in vivo PET imaging study.

EXAMPLE. Radiochemical synthesis of DUPA-C-NETA based theranostics.

EXAMPLE. Preparation of the NOTA Derivatives. Bifunctional conjugates,also referred to as theranotics, are described herein. Compoundsdescribed herein can tightly chelate both radionuclides such as ¹⁸F and⁶⁸Ga for PET imaging, and radionuclides ¹⁷⁷Lu and ⁹⁰Y for radiotherapy.C-NETA, a NOTA derivative, has been reported to chelate Al¹⁸F with abouttwice the efficiency (87%) of NOTA.[17] Moreover, C-NETA also reportedlychelates the commonly used radiotherapeutic nuclides, such as ¹⁷⁷Lu and⁹⁰Y, with high labeling efficiency.[18] Thus, it is appreciated herein,that C-NETA is useful as a bifunctional chelator that can be used forboth PET imaging and radiotherapy, where the radionuclide is a metal ormetal halide, such as Al¹⁸F, ⁶⁸Ga, ¹⁷⁷Lu or ⁹⁰Y.

EXAMPLE. A PyBOP promoted coupling between QC04018 and QC08008, followedby deprotection of tert-butyl ester with TFA provides DUPA-C-NETA.DUPA-C-NETA is used to evaluate the labeling efficiency to Al¹⁸F, ¹⁷⁷Luand ⁹⁰Y, and evaluate the in vivo PET imaging and radiotherapy.

METHOD EXAMPLES

EXAMPLE. The specificity of the radionuclide containing conjugatesbinding to FR is evaluated against KB xenografts homogenates and Cal51xenografts homogenates. Concentration dependent binding was evaluatedfor ¹⁸F-AIF-QC07017 and ¹⁸F-AIF-QC07043, and separated into specific andnon-specific binding. Significant non-specific binding was not observedin KB homogenates. Minor non-specific binding was observed in Cal51homogenates, with a specific/non-specific binding ratio of >3:1 at allconcentrations up to about 30 nM for ¹⁸F-AIF-QC07017, and aspecific/non-specific binding ratio of >2:1 at all concentrations up toabout 20 nM for ¹⁸F-AIF-QC07043. Minor non-specific binding was observedin A549 homogenates, with a specific/non-specific binding ratio of >2:1at all concentrations up to about 10 nM for ¹⁸F-AIF-QC07043. Scatchardanalyses were also performed. Displaceable and saturable binding of¹⁸F-AIF-QC07017 in human tumor xenografts (KB and Cal51) by selfcompetition was observed. Both ¹⁸F-AIF-QC07017 and ¹⁸F-AIF-QC07043 boundone site with high affinity in all cell xenografts. The high ratio ofBmax/Kd indicated a high specific binding affinity to KB xenografts.Moderate binding affinity was observed for Cal51 xenografts, and thelowest binding affinity was observed for A549 xenografts. Without beingbound by theory, it is believed herein that the moderate expression ofFR in Cal51 xenografts accounts for the lower binding affinity.

Binding affinities of ¹⁸F-AIF-QC07017 (2) to FR in KB and Cal51 tumorcrude homogenate.

Folate-NOTA-A118F (2) Bmax, nM* Kd, nM Bmax/Kd KB xenografts 5110.7 >700 Cal51 xenografts 36 1.1 >30

Binding affinities of ¹⁸F-AIF-QC07043 to FR in KB and Cal51 tumor crudehomogenate.

FA-PEG12-NOTA-A118F Bmax, nM* Kd, nM Bmax/Kd KB xenografts 241 0.4 603Cal51 xenografts 13 1.2 11

EXAMPLE. μPET imaging was performed on nude mice bearing KB tumorxenografts under baseline and competed conditions to evaluate the invivo binding specificity of ¹⁸F-AIF-QC07017 (2) to FR. Nude mice bearingKB tumor xenografts on their left shoulder were injected with 0.30-0.40mCi (2). The competed group received 100 μg of folic acid 10 min beforethe i.v. injection of (2), and the treatment group was injected with acorresponding volume of phosphate buffer. Time course inspection of PETimages obtained at various time points revealed that the data acquiredin 60-90 min post tracer injection gave the best visual PET imaging. TheKB tumors were clearly visualized in the treated group, whereus theuptake of (2) was completely inhibited by competing with folic acid,supporting a high specificity of (2) binding to FR in vivo. Withoutbeing bound by theory, it is believed herein that the high radioactivityfound in kidneys was due to the uptake mediated by FR that is expressedin the proximal tubule cells in kidneys and the potential accumulationof radiotracer via renal excretion, which was further supported by thebiodistribution studies described herein. With the exception of theliver, significant uptake in other organs was not observed. Asignificant blocking effect in liver uptake was observed in undercompeted conditions.

EXAMPLE. Ex vivo biodistribution study of compounds described hereinunder both baseline and competed conditions in nude mice bearing KBtumor xenografts on their left shoulder demonstrates a high and specificuptake in FR(+) tumors. Radiotracer levels of ¹⁸F-AIF-QC07017 and¹⁸F-AIF-QC07043 were determined in whole blood, plasma, heart, kidney,liver, lung, muscle, spleen, KB xenograft tumor tissues and A549xenograft tumor tissues (FIG. 1A, FIG. 1B, and FIG. 1C). The highestsignal was observed in the kidneys. Accumulation was observed to asubstantially less extent in the liver. Without being bound by theory,it is believed herein that the highest accumulation of radioactivity inkidneys, along with the relative low uptake of radiotracer in thehepatobiliary system i.e. liver, bile and intestine/feces supports thatrenal elimination is the predominant excretion pathway. Except for thekidneys, accumulation in KB xenograft tumor tissues was greatest, andsignificantly greater than in the liver. Accumulation in A549 xenografttumor tissues was comparable to the liver. Accumulation in both KBxenograft tumor tissues and A549 xenograft tumor tissues was blockedunder competition conditions with folic acid (FIG. 2A and FIG. 2B). TheFR specificity of ¹⁸F-AIF-QC07017 and ¹⁸F-AIF-QC07043 was comparable toetarfolatide (EC20), a compound in clinical trials, in both KB xenografttumor tissues and A549 xenograft tumor tissues.

Uptake in KB Xenografts

Uptake under Uptake competed conditions (SUV) (SUV) Example (±SEM)(±SEM) ¹⁸F-AIF-QC07017 2.84 ± 0.76 0.34 ± 0.02 ¹⁸F-AIF-QC07043 2.33 ±0.13 0.41 ± 0.06 ^(99m)Tc-EC20 2.75 ± 0.14 0.43 ± 0.05 P values:^(99m)Tc-EC20 vs ¹⁸F-AIF-QC07043, p = 0.15; ¹⁸F-AIF-QC07017 vs¹⁸F-AIF-QC07043, p = 0.48; EC20 vs ¹⁸F-AIF-QC07017, p = 0.85.

Uptake in A549 Xenografts

Uptake under Uptake competed conditions (SUV) (SUV) Example (±SEM)(±SEM) ¹⁸F-AIF-QC07017 0.64 ± 0.16 0.03 ± 0.01 ¹⁸F-AIF-QC07043 0.53 ±0.06 0.04 ± 0.01 ^(99m)Tc-EC20 0.71 ± 0.09 0.08 ± 0.02 P values:^(99m)Tc-EC20 vs ¹⁸F-AIF-QC07043, p = 0.13; ¹⁸F-AIF-QC07017 vs¹⁸F-AIF-QC07043, p = 0.50; ^(99m)Tc-EC20 vs ¹⁸F-AIF-QC07017, p = 0.74

EXAMPLE. In vitro evaluation of DUPA-EAOA-Phe-Phe-NOTA-⁶⁸Ga radiotracer(⁶⁸Ga-QC08009). ⁶⁷Ga has a longer half life than ⁶⁸Ga (about 3.3 daysversus about 68 minutes, respectively). Thus, ⁶⁷Ga is used as asurrogate of ⁶⁸Ga for in vitro evaluation of Kd values and tissueimaging. It is to be understood that the in vitro evaluation of Kdvalues and tissue imaging observed for ⁶⁷Ga is predictive of ⁶⁸Ga.DUPA-EAOA-Phe-Phe-NOTA-⁶⁷Ga (67Ga-NOTA-LC-PSMA2) was prepared in nearlyquantitative radiochemical yield. In vitro study in both the PSMA(−)cell line (PC3) and the PSMA(+) cell lines (LnCaP and PIP-PC3) revealeda PSMA mediated high and specific uptake with a Kd=8.45±2.16 nM. PC3 isa PSMA (−) cell line; LnCap is a PSMA (+) cell line; and PIP-PC3 is atransfect cell line with higher PSMA expression. Uptake of ⁶⁸Ga-QC08009by PC3 cells was minimal, and did not change when competed. Uptake of⁶⁸Ga-QC08009 by LnCaP and PIP-PC3 was substantial, with PIP-PC3 cellsshowing the highest uptake. In both cases, Uptake of ⁶⁸Ga-QC08009 byLnCaP and PIP-PC3 is blocked by competing ligand. Compared to⁶⁷Ga-DKFZ-PSMA11, an imaging agent in clinical trials,⁶⁷Ga-NOTA-LC-PSMA2 demonstrated superior binding to PSMA(+) prostatecancer tissues. EXAMPLE. In vivo PET imaging and BioD study ofDUPA-EAOA-Phe-Phe-NOTA-⁶⁸Ga radiotracer (⁶⁸Ga-QC08009). In vivomicro-PET/CT scan with ⁶⁸Ga-NOTA-LC-PSMA2 radiotracer in mice carryingPSMA (+) LnCaP xenografts showed 4.29% ID uptake in PSMA(+) tumor. At 1hour post-injection, most of the radiotracer was found in bladder.Without being bound by theory, it is believed herein that the datasupport that the primary elimination pathway is in urine. In addition,compared to other tissues, minor accumulation of radiotracer wasobserved in the kidneys. Without being bound by theory, it is believedherein that the relatively high PSMA expression in mouse kidneys,compared to other tissues, accounts at least in part for the minoraccumulation of ⁶⁸Ga-NOTA-LC-PSMA2 radiotracer in kidneys.

1. A conjugate of the formulaB-L-P or a pharmaceutically acceptable salt thereof, wherein B is aradical of a targeting agent selected from vitamin receptor bindingligands, PSMA binding ligands, and PSMA inhibitors, L is a divalentlinker, and P is a radical comprising a formula chosen from the groupconsisting of:


2. The conjugate of claim 1 comprising folate-Asp.
 3. The conjugate ofclaim 1 comprising folate-Arg.
 4. The conjugate of claim 1, wherein thedivalent linker L comprises a polypeptide comprising phenylalanine,lysine, arginine, or aspartic acid, or a combination thereof.
 5. Theconjugate of claim 1 wherein the divalent linker L does not include adiradical of the formula *NH—(CH₂)₂—NH*.
 6. The conjugate of claim 1,wherein P comprises:

or a derivative thereof comprising a chelated metal.
 7. The conjugate ofclaim 1 comprising folate-PEG.
 8. The conjugate of claim 1, wherein Pcomprises:

or a derivative thereof comprising a chelated metal.
 9. The conjugate ofclaim 1, wherein P comprises:

or a derivative thereof comprising a chelated metal.
 10. The conjugateof claim 1 wherein the targeting agent is a radical of a PSMA bindingligand or PSMA inhibitor.
 11. The conjugate of claim 10 comprising theformula

where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or

where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or

where W is O or S.
 12. The conjugate of claim 10 wherein the linkercomprises a polypeptide comprising phenylalanine, lysine, arginine, oraspartic acid, or a combination thereof.
 13. The conjugate of claim 1,wherein P comprises:

or a derivative thereof comprising a chelated metal.
 14. The conjugateof claim 10 comprising the formula

where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.15. The conjugate of claim 10 wherein the divalent linker L comprisesthe formula


16. The conjugate of claim 10 wherein the divalent linker L comprisesthe formula


17. The conjugate of claim 1, wherein P comprises:

or a derivative thereof comprising a chelated metal.
 18. The conjugateof claim 1, further comprising a positron-emitting radionuclide orradionuclide containing group.
 19. The conjugate of claim 18 wherein theradionuclide is a radiotherapy agent.
 20. The conjugate of claim 18,wherein the positron-emitting radionuclide or radionuclide containinggroup is selected from the group consisting of ⁸⁹Zr, ⁴⁵Ti, ⁵¹Mn, ⁶⁴Cu,⁶¹Cu, ⁶³Zn, ⁸²Rb, ⁶⁸Ga, ⁶⁶Ga, ¹¹C, ¹³N, ¹⁵O, ¹²⁴I, ³⁴Cl, ¹⁸F, and ¹⁹F.